Novel glyphosate N-acetyltransferase (GAT) genes

ABSTRACT

Novel proteins are provided herein, including proteins capable of catalyzing the acetylation of glyphosate and other structurally related proteins. Also provided are novel polynucleotides capable of encoding these proteins, compositions that include one or more of these novel proteins and/or polynucleotides, recombinant cells and transgenic plants comprising these novel compounds, diversification methods involving the novel compounds, and methods of using the compounds. Some of the novel methods and compounds provided herein can be used to render an organism, such as a plant, resistant to glyphosate.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to and benefit of U.S.Provisional Patent Application No. 60/377,719 filed Apr. 30, 2002, thedisclosure of which is incorporated herein by reference in its entiretyfor all purposes and U.S. Provisional Patent Application No. 60/377,175filed May 1, 2002, the disclosure of which is incorporated herein byreference in its entirety for all purposes; and this application is acontinuation-in-part of and claims priority to and benefit of co-pendingU.S. application Ser. No. 10/004,357 filed on Oct. 29,2001, thedisclosure of which is incorporated herein by reference in its entiretyfor all purposes, which claims priority to and benefit of U.S.Provisional Application No. 60/244,385 filed on Oct. 30, 2000, thedisclosure of which is incorporated herein by reference in its entiretyfor all purposes.

COPYRIGHT NOTIFICATION PURSUANT TO 37 C.F.R. § 1.71 (E)

[0002] A portion of the disclosure of this patent document containsmaterial which is subject to copyright protection. The copyright ownerhas no objection to the facsimile reproduction by anyone of the patentdocument or the patent disclosure, as it appears in the Patent andTrademark Office patent file or records, but otherwise reserves allcopyright rights whatsoever.

BACKGROUND OF THE INVENTION

[0003] Crop selectivity to specific herbicides can be conferred byengineering genes into crops which encode appropriate herbicidemetabolizing enzymes. In some cases these enzymes, and the nucleic acidsthat encode them, originate in a plant. In other cases, they are derivedfrom other organisms, such as microbes. See, e.g., Padgette et al.(1996) “New weed control opportunities: Development of soybeans with aRound UP Ready™ gene” in Herbicide-Resistant Crops (Duke, ed.), pp54-84,CRC Press, Boca Raton; and Vasil (1996) “Phosphinothricin-resistantcrops” in Herbicide-Resistant Crops (Duke, ed.), pp85-91. Indeed,transgenic plants have been engineered to express a variety of herbicidetolerance/metabolizing genes, from a variety of organisms. For example,acetohydroxy acid synthase, which has been found to make plants thatexpress this enzyme resistant to multiple types of herbicides, has beenintroduced into a variety of plants (see, e.g., Hattori et al. (1995)Mol Gen Genet 246:419). Other genes that confer tolerance to herbicidesinclude: a gene encoding a chimeric protein of rat cytochrome P4507A1and yeast NADPH-cytochrome P450 oxidoreductase (Shiota et al. (1994)Plant Physiol. 106:17), genes for glutathione reductase and superoxidedismutase (Aono et al. (1995) Plant Cell Physiol. 36:1687, and genes forvarious phosphotransferases (Datta et al. (1992) Plant Mol Biol 20:619).

[0004] One herbicide which is the subject of much investigation in thisregard is N-phosphonomethylglycine, commonly referred to as glyphosate.Glyphosate is the top selling herbicide in the world, with salesprojected to reach $5 billion by 2003. It is a broad spectrum herbicidethat kills both broadleaf and grass-type plants. A successful mode ofcommercial level glyphosate resistance in transgenic plants is byintroduction of a modified Agrobacterium CP45-enolpyruvylshikimate-3-phosphate synthase (hereinafter referred to asEPSP synthase or EPSPS) gene. The transgene is targeted to thechloroplast where it is capable of continuing to synthesize EPSPsynthase from phosphoenolpyruvic acid (PEP) and shikimate-3-phosphate inthe presence of glyphosate. In contrast, the native EPSP synthase isinhibited by glyphosate. Without the transgene, plants sprayed withglyphosate quickly die due to inhibition of EPSP synthase which haltsthe downstream pathway needed for aromatic amino acid, hormone, andvitamin biosynthesis. The CP4 glyphosate-resistant soybean transgenicplants are marketed, e.g., by Monsanto under the name “Round UP Ready™.”

[0005] In the environment, the predominant mechanism by which glyphosateis degraded is through soil microflora metabolism. The primarymetabolite of glyphosate in soil has been identified asaminomethylphosphonic acid (AMPA), which is ultimately converted intoammonia, phosphate and carbon dioxide. The proposed metabolic schemethat describes the degradation of glyphosate in soil through the AMPApathway is shown in FIG. 8. An alternative metabolic pathway for thebreakdown of glyphosate by certain soil bacteria, the sarcosine pathway,occurs via initial cleavage of the C—P bond to give inorganic phosphateand sarcosine, as depicted in FIG. 9.

[0006] Another successful herbicide/transgenic crop package isglufosinate (phosphinothricin) and the Liberty Link™ trait marketed,e.g., by Aventis. Glufosinate is also a broad spectrum herbicide. Itstarget is the glutamate synthase enzyme of the chloroplast. Resistantplants carry the bar gene from Streptomyces hygroscopicus and achieveresistance by the N-acetylation activity of bar, which modifies anddetoxifies glufosinate.

[0007] An enzyme capable of acetylating the primary amine of AMPA isreported in PCT Application No. WO00/29596. The enzyme was not describedas being able to acetylate a compound with a secondary amine (e.g.,glyphosate).

[0008] While a variety of herbicide resistance strategies are availableas noted above, additional approaches would have considerable commercialvalue. The present invention provides novel polynucleotides andpolypeptides for conferring herbicide tolerance, as well as numerousother benefits as will become apparent during review of the disclosure.

SUMMARY OF THE INVENTION

[0009] The present invention provides methods and reagents for renderingan organism, such as a plant, resistant to glyphosate by one or more ofthe embodiments described below.

[0010] One embodiment of the invention provides novel polypeptidesreferred to herein as glyphosate-N-acetyltransferase (“GAT”)polypeptides. GAT polypeptides are characterized by their structuralsimilarity to one another, e.g., in terms of sequence similarity whenthe GAT polypeptides are aligned with one another. GAT polypeptides ofthe present invention possess glyphosate-N-acetyl transferase activity,i.e., the ability to catalyze the acetylation of glyphosate. These GATpolypeptides transfer the acetyl group from acetyl CoA to the N ofglyphosate. In addition, some GAT polypeptides transfer the propionylgroup of propionyl CoA to the N of glyphosate. Some GAT polypeptides arealso capable of catalyzing the acetylation of glyphosate analogs and/orglyphosate metabolites, e.g., aminomethylphosphonic acid. Exemplary GATpolypeptides correspond to SEQ ID NO:6-10, 263-514, 568-619, 621, 623,625, 627, 629, 631, 633, 635, 637, 639, 641, 643, 645, 647, 649, 651,653, 655, 657, 659, 661, 663, 665, 667, 669, 671, 673, 675, 677, 679,681, 683, 685, 687, 689, 691, 693, 695, 697, 699, 701, 703, 705, 707,709, 711, 713, 715, 717, 719, 721, 723, 725, 727, 729, 731, 733, 735,737, 739, 741, 743, 745, 747, 749, 751, 753, 755, 757, 759, 761, 763,765, 767, 769, 771, 773, 775, 777, 779, 781, 783, 785, 787, 789, 791,793, 795, 797, 799, 801, 803, 805, 807, 809, 811, and 813.

[0011] Also provided are novel polynucleotides referred to herein as GATpolynucleotides, e.g., SEQ ID NO:1-5, 11-262, 516-567, 620, 622, 624,626, 628, 630, 632, 634, 636, 638, 640, 642, 644, 646, 648, 650, 652,654, 656, 658, 660, 662, 664, 666, 668, 670, 672, 674, 676, 678, 680,682, 684, 686, 688, 690, 692, 694, 696, 698, 700, 702, 704, 706, 708,710, 712, 714, 716, 718, 720, 722, 724, 726, 728, 730, 732, 734, 736,738, 740, 742, 744, 746, 748, 750, 752, 754, 756, 758, 760, 762, 764,766, 768, 770, 772, 774, 776, 778, 780, 782, 784, 786, 788, 790, 792,794, 796, 798, 800, 802, 804, 806, 808, 810, and 812. GATpolynucleotides are characterized by their ability to encode GATpolypeptides. In some embodiments of the invention, a GAT polynucleotideis engineered for better plant expression by replacing one or moreparental codons with a synonymous codon that is preferentially used inplants relative to the parental codon. In other embodiments, a GATpolynucleotide is modified by the introduction of a nucleotide sequenceencoding an N-terminal chloroplast transit peptide.

[0012] GAT polypeptides, GAT polynucleotides and glyphosate-N-acetyltransferase activity are described in more detail below. The inventionfurther includes certain fragments of the GAT polypeptides and GATpolynucleotides described herein.

[0013] The invention includes non-native variants of the polypeptidesand polynucleotides described herein, wherein one or more amino acids ofthe encoded polypeptide have been mutated.

[0014] In certain preferred embodiments, the GAT polypeptides of thepresent invention are characterized as follows. When optimally alignedwith a reference amino acid sequence selected from the group consistingof SEQ ID NO:6-10, 263-514, 568-619, 621, 623, 625, 627, 629, 631, 633,635, 637, 639, 641, 643, 645, 647, 649, 651, 653, 655, 657, 659, 661,663, 665, 667, 669, 671, 673, 675, 677, 679, 681, 683, 685, 687, 689,691, 693,695,697,699,701,703,705,707,709,711,713,715,717,719,721,723,725,727, 729, 731, 733, 735, 737, 739, 741,743,745,747,749, 751,753,755, 757,759, 761,763, 765, 767, 769, 771, 773, 775, 777, 779, 781,783, 785, 787, 789, 791, 793, 795, 797, 799, 801, 803, 805, 807, 809,811, and 813, one or more of the following positions conform to thefollowing restrictions: (a) at position 75, the amino acid is selectedfrom the group consisting of B1, Z1, M or V; (b) at position 58, theamino acid is selected from the group consisting of B2, Z3, Z4, Z6, K,P, Q or R; (c) at position 47, the amino acid is selected from the groupconsisting of B2, Z4, Z6, R and G; (d) at position 45, the amino acid isselected from the group consisting of B1, Z2, F or Y; (e) at position91, the amino acid is selected from the group consisting of B1, Z1, L, Vor I; (f) at position 105, the amino acid is selected from B1, Z1, I, Mor L; (g) at position 129, the amino acid is selected from the groupconsisting of B1, Z1, I or V; and (h) at position 89, the amino acid isselected from the group consisting of B2, Z3, Z6, G, T or S, wherein B1is an amino acid selected from the group consisting of A, I, L, M, F, W,Y, and V; B2 is an amino acid selected from the group consisting of R,N, D, C, Q, E, G, H, K, P, S, and T; Z1 is an amino acid selected fromthe group consisting of A, I, L, M, and V; Z2 is an amino acid selectedfrom the group consisting of F, W, and Y; Z3 is an amino acid selectedfrom the group consisting of N, Q, S, and T; Z4 is an amino acidselected from the group consisting of R, H, and K; Z5 is an amino acidselected from the group consisting of D and E; and Z6 is an amino acidselected from the group consisting of C, G, and P.

[0015] The invention further provides a nucleic acid constructcomprising a polynucleotide of the invention. The construct can be avector, such as a plant transformation vector. In some aspects a vectorof the invention will comprise a T-DNA sequence. The construct canoptionally include a regulatory sequence (e.g., a promoter) operablylinked to a GAT polynucleotide, where the promoter is heterologous withrespect to the polynucleotide and effective to cause sufficientexpression of the encoded polypeptide to enhance the glyphosatetolerance of a plant cell transformed with the nucleic acid construct.

[0016] In some aspects of the invention, a GAT polynucleotide functionsas a selectable marker, e.g., in a plant, bacteria, actinomycetes,yeast, algae or other fungi. For example, an organism that has beentransformed with a vector including a GAT polynucleotide selectablemarker can be selected based on its ability to grow in the presence ofglyphosate. A GAT marker gene can be used for selection or screening fortransformed cells expressing the gene.

[0017] The invention further provides vectors with stacked traits, i.e.,vectors that encode a GAT polypeptide and that also include a secondpolynucleotide sequence encoding a second polypeptide that confers adetectable phenotypic trait upon a cell or organism expressing thesecond polypeptide at an effective level, for example disease resistanceor pest resistance. The detectable phenotypic trait can also function asa selectable marker, e.g., by conferring herbicide resistance or byproviding some sort of visible marker.

[0018] In one embodiment, the invention provides a compositioncomprising two or more polynucleotides of the invention. Preferably, theGAT polynucleotides encode GAT polypeptides having different kineticparameters, i.e., a GAT variant having a lower K_(m) can be combinedwith one having a higher k_(cat). In a further embodiment, the differentGAT polynucleotides may be coupled to a chloroplast transit sequence orother signal sequence thereby providing GAT polypeptide expression indifferent cellular compartments, organelles or secretion of one or moreof the GAT polypeptides.

[0019] Accordingly, compositions containing two or more GATpolynucleotides or encoded polypeptides are a feature of the invention.In some cases, these compositions are libraries of nucleic acidscontaining, e.g., at least 3 or more such nucleic acids. Compositionsproduced by digesting the nucleic acids of the invention with arestriction endonuclease, a DNAse or an RNAse, or otherwise fragmentingthe nucleic acids, e.g., mechanical shearing, chemical cleavage, etc.,are also a feature of the invention, as are compositions produced byincubating a nucleic acid of the invention with deoxyribonucleotidetriphosphates and a nucleic acid polymerase, such as a thermostablenucleic acid polymerase.

[0020] Cells transduced by a vector of the invention, or which otherwiseincorporate a nucleic acid of the invention, are an aspect of theinvention. In a preferred embodiment, the cells express a polypeptideencoded by the nucleic acid.

[0021] In some embodiments, the cells incorporating the nucleic acids ofthe invention are plant cells. Transgenic plants, transgenic plant cellsand transgenic plant explants incorporating the nucleic acids of theinvention are also a feature of the invention. In some embodiments, thetransgenic plants, transgenic plant cells or transgenic plant explantsexpress an exogenous polypeptide with glyphosate-N-acetyltransferaseactivity encoded by the nucleic acid of the invention. The inventionalso provides transgenic seeds produced by the transgenic plants of theinvention.

[0022] The invention further provides transgenic plants, transgenicplant cells, transgenic plant explants, or transgenic seeds havingenhanced tolerance to glyphosate due to the expression of a polypeptidewith glyphosate-N-acetyltransferase activity and a polypeptide thatimparts glyphosate tolerance by another mechanism, such as, aglyphosate-tolerant 5-enolpyruvylshikimate-3-phosphate synthase and/or aglyphosate-tolerant glyphosate oxido-reductase. In a further embodiment,the invention provides transgenic plants or transgenic plant explantshaving enhanced tolerance to glyphosate, as well as tolerance to anadditional herbicide due to the expression of a polypeptide withglyphosate-N-acetyltransferase activity, a polypeptide that impartsglyphosate tolerance by another mechanism, such as, aglyphosate-tolerant 5-enolpyruvylshikimate-3-phosphate synthase and/or aglyphosate-tolerant glyphosate oxido-reductase and a polypeptideimparting tolerance to the additional herbicide, such as, a mutatedhydroxyphenylpyruvatedioxygenase, a sulfonamide-tolerant acetolactatesynthase, a sulfonamide-tolerant acetohydroxy acid synthase, animidazolinone-tolerant acetolactate synthase, an imidazolinone-tolerantacetohydroxy acid synthase, a phosphinothricin acetyl transferase and amutated protoporphyrinogen oxidase.

[0023] The invention also provides transgenic plants, transgenic plantcells, transgenic plant explants, or transgenic seeds having enhancedtolerance to glyphosate, as well as tolerance to an additional herbicidedue to the expression of a polypeptide withglyphosate-N-acetyltransferase activity and a polypeptide impartingtolerance to the additional herbicide, such as, a mutatedhydroxyphenylpyruvatedioxygenase, a sulfonamide-tolerant acetolactatesynthase, a sulfonamide-tolerant acetohydroxy acid synthase, animidazolinone-tolerant acetolactate synthase, an imidazolinone-tolerantacetohydroxy acid synthase, a phosphinothricin acetyl transferase and amutated protoporphyrinogen oxidase.

[0024] Methods of producing the polypeptides of the invention byintroducing the nucleic acids encoding them into cells and thenexpressing and recovering them from the cells or culture medium are afeature of the invention. In preferred embodiments, the cells expressingthe polypeptides of the invention are transgenic plant cells.

[0025] Polypeptides that are specifically bound by a polyclonal antiserathat reacts against an antigen derived from SEQ ID NO:6-10, 263-514,568-619, 621, 623, 625, 627, 629, 631, 633, 635, 637, 639, 641, 643,645, 647, 649, 651, 653, 655, 657, 659, 661, 663, 665, 667, 669, 671,673, 675, 677, 679, 681, 683, 685, 687, 689, 691, 693, 695, 697, 699,701,703,705,707,709, 711, 713,715,717,719,721, 723,725,727,729,731,733,735, 737, 739, 741, 743, 745, 747, 749, 751, 753, 755, 757, 759,761, 763, 765, 767, 769, 771, 773, 775, 777, 779, 781, 783, 785, 787,789, 791, 793, 795, 797, 799, 801, 803, 805, 807, 809, 811, and 813 butnot to a naturally occurring related sequence, e.g., such as a peptiderepresented by a subsequence of those of GenBank accession numberCAA70664, as well as antibodies which are produced by administering anantigen derived from any one or more of SEQ ID NO:6-10, 263-514,568-619, 621, 623, 625, 627, 629, 631, 633, 635, 637, 639, 641, 643,645, 647, 649, 651, 653, 655, 657, 659, 661, 663, 665, 667, 669, 671,673, 675, 677, 679, 681, 683, 685, 687, 689, 691, 693, 695, 697, 699,701, 703, 705, 707, 709, 711,713,715,717,719, 721,723,725,727,729,731,733,735,737,739, 741,743,745, 747, 749, 751, 753, 755, 757, 759,761, 763, 765, 767, 769, 771, 773, 775, 777, 779, 781, 783, 785, 787,789, 791, 793, 795, 797, 799, 801, 803, 805, 807, 809, 811, and 813and/or which bind specifically to such antigens and which do notspecifically bind to a naturally occurring polypeptide corresponding tothose of GenBank accession number CAA70664, are all features of theinvention.

[0026] Another aspect of the invention relates to methods ofpolynucleotide diversification to produce novel GAT polynucleotides andpolypeptides by recombining or mutating the nucleic acids of theinvention in vitro or in vivo. In an embodiment, the recombinationproduces at least one library of recombinant GAT polynucleotides. Thelibraries so produced are embodiments of the invention, as are cellscomprising the libraries. Furthermore, methods of producing a modifiedGAT polynucleotide by mutating a nucleic acid of the invention areembodiments of the invention. Recombinant and mutant GAT polynucleotidesand polypeptides produced by the methods of the invention are alsoembodiments of the invention.

[0027] In some aspects of the invention, diversification is achieved byusing recursive recombination, which can be accomplished in vitro, invivo, in silico, or a combination thereof Some examples ofdiversification methods described in more detail below are familyshuffling methods and synthetic shuffling methods. The inventionprovides methods for producing a glyphosate resistant transgenic plantor plant cell that involve transforming a plant or plant cell with apolynucleotide encoding a glyphosate-N-acetyltransferase, and optionallyregenerating a transgenic plant from the transformed plant cell. In someaspects the polynucleotide is a GAT polynucleotide, optionally a GATpolynucleotide derived from a bacterial source. In some aspects of theinvention, the method can comprise growing the transformed plant orplant cell in a concentration of glyphosate that inhibits the growth ofa wild-type plant of the same species without inhibiting the growth ofthe transformed plant. The method can comprise growing the transformedplant or plant cell or progeny of the plant or plant cell in increasingconcentrations of glyphosate and/or in a concentration of glyphosatethat is lethal to a wild-type plant or plant cell of the same species. Aglyphosate resistant transgenic plant produced by this method can bepropagated, for example by crossing it with a second plant, such that atleast some progeny of the cross display glyphosate tolerance.

[0028] The invention further provides methods for selectivelycontrolling weeds in a field containing a crop that involve planting thefield with crop seeds or plants which are glyphosate-tolerant as aresult of being transformed with a gene encoding a glyphosateN-acetyltransferase, and applying to the crop and weeds in the field asufficient amount of glyphosate to control the weeds withoutsignificantly affecting the crop.

[0029] The invention further provides methods for controlling weeds in afield and preventing the emergence of glyphosate resistant weeds in afield containing a crop which involve planting the field with crop seedsor plants that are glyphosate tolerant as a result of being transformedwith a gene encoding a glyphosate-N-acetyltransferase and a geneencoding a polypeptide imparting glyphosate tolerance by anothermechanism, such as, a glyphosate-tolerant5-enolpyruvylshikimate-3-phosphate synthase and/or a glyphosate-tolerantglyphosate oxido-reductase and applying to the crop and the weeds in thefield a sufficient amount of glyphosate to control the weeds withoutsignificantly affecting the crop.

[0030] In a further embodiment the invention provides methods forcontrolling weeds in a field and preventing the emergence of herbicideresistant weeds in a field containing a crop which involve planting thefield with crop seeds or plants that are glyphosate tolerant as a resultof being transformed with a gene encoding aglyphosate-N-acetyltransferase, a gene encoding a polypeptide impartingglyphosate tolerance by another mechanism, such as, aglyphosate-tolerant 5-enolpyruvylshikimate-3-phosphate synthase and/or aglyphosate-tolerant glyphosate oxido-reductase and a gene encoding apolypeptide imparting tolerance to an additional herbicide, such as, amutated hydroxyphenylpyruvatedioxygenase, a sulfonamide-tolerantacetolactate synthase, a sulfonamide-tolerant acetohydroxy acidsynthase, an imidazolinone-tolerant acetolactate synthase, animidazolinone-tolerant acetohydroxy acid synthase, a phosphinothricinacetyl transferase and a mutated protoporphyrinogen oxidase and applyingto the crop and the weeds in the field a sufficient amount of glyphosateand an additional herbicide, such as, a hydroxyphenylpyruvatedioxygenaseinhibitor, sulfonamide, imidazolinone, bialaphos, phosphinothricin,azafenidin, butafenacil, sulfosate, glufosinate, and a protox inhibitorto control the weeds without significantly affecting the crop.

[0031] The invention further provides methods for controlling weeds in afield and preventing the emergence of herbicide resistant weeds in afield containing a crop which involve planting the field with crop seedsor plants that are glyphosate tolerant as a result of being transformedwith a gene encoding a glyphosate-N-acetyltransferase and a geneencoding a polypeptide imparting tolerance to an additional herbicide,such as, a mutated hydroxyphenylpyruvatedioxygenase, asulfonamide-tolerant acetolactate synthase, a sulfonamide-tolerantacetohydroxy acid synthase, an imidazolinone-tolerant acetolactatesynthase, an imidazolinone-tolerant acetohydroxy acid synthase, aphosphinothricin acetyl transferase and a mutated protoporphyrinogenoxidase and applying to the crop and the weeds in the field a sufficientamount of glyphosate and an additional herbicide, such as, ahydroxyphenylpyruvatedioxygenase inhibitor, sulfonamide, imidazolinone,bialaphos, phosphinothricin, azafenidin, butafenacil, sulfosate,glufosinate, and a protox inhibitor to control the weeds withoutsignificantly affecting the crop.

[0032] The invention further provides methods for producing agenetically transformed plant that is tolerant to glyphosate thatinvolve inserting into the genome of a plant cell a recombinant,double-stranded DNA molecule comprising: (i) a promoter which functionsin plant cells to cause the production of an RNA sequence;(ii) astructural DNA sequence that causes the production of an RNA sequencewhich encodes a GAT; and (iii) a 3′ non-translated region whichfunctions in plant cells to cause the addition of a stretch ofpolyadenyl nucleotides to the 3′ end of the RNA sequence; where thepromoter is heterologous with respect to the structural DNA sequence andadapted to cause sufficient expression of the encoded polypeptide toenhance the glyphosate tolerance of a plant cell transformed with theDNA molecule; obtaining a transformed plant cell; and regenerating fromthe transformed plant cell a genetically transformed plant which hasincreased tolerance to glyphosate.

[0033] The invention further provides methods for producing a crop thatinvolve growing a crop plant that is glyphosate-tolerant as a result ofbeing transformed with a gene encoding a glyphosate N-acetyltransferase,under conditions such that the crop plant produces a crop; andharvesting a crop from the crop plant. These methods often includeapplying glyphosate to the crop plant at a concentration effective tocontrol weeds. Exemplary crop plants include cotton, corn, and soybean.

[0034] The invention also provides computers, computer readable mediumand integrated systems, including databases that are composed ofsequence records including character strings corresponding to SEQ IDNO:1-514 and 516-813. Such integrated systems optionally include, one ormore instruction set for selecting, aligning, translating,reverse-translating or viewing any one or more character stringscorresponding to SEQ ID NO: 1-514 and 516-813, with each other and/orwith any additional nucleic acid or amino acid sequence.

BRIEF DESCRIPTION OF THE FIGURES

[0035]FIG. 1 depicts the N-acetylation of glyphosate catalyzed by aglyphosate-N-acetyltransferase (“GAT”).

[0036]FIG. 2 illustrates mass spectroscopic detection ofN-acetylglyphosate produced by an exemplary Bacillus culture expressinga native GAT activity.

[0037]FIG. 3 is a table illustrating the relative identity between GATsequences isolated from different strains of bacteria and yitI fromBacillus subtilis.

[0038]FIG. 4 is a map of the plasmid pMAXY2120 for expression andpurification of the GAT enzyme from E. coli cultures.

[0039]FIG. 5 is a mass spectrometry output showing increasedN-acetylglyphosate production over time in a typical GAT enzyme reactionmix.

[0040]FIG. 6 is a plot of the kinetic data of a GAT enzyme from which aK_(M) of 2.9 mM for glyphosate was calculated.

[0041]FIG. 7 is a plot of the kinetic data taken from the data of FIG. 6from which a K_(M) of 2 μM was calculated for Acetyl CoA.

[0042]FIG. 8 is a scheme that describes the degradation of glyphosate insoil through the AMPA pathway.

[0043]FIG. 9 is a scheme that describes the sarcosine pathway ofglyphosate degradation.

[0044]FIG. 10 is the BLOSUM62 matrix.

[0045]FIG. 11 is a map of the plasmid pMAXY2190.

[0046]FIG. 12 depicts a T-DNA construct with gat selectable marker.

[0047]FIG. 13 depicts a yeast expression vector with gat selectablemarker.

[0048]FIG. 14 illustrates effect of glyphosate on plant height attasseling.

DETAILED DISCUSSION

[0049] The present invention relates to a novel class of enzymesexhibiting N-acetyltransferase activity. In one aspect, the inventionrelates to a novel class of enzymes capable of acetylating glyphosateand glyphosate analogs, e.g., enzymes possessingglyphosate-N-acetyltransferase (“GAT”) activity. Such enzymes arecharacterized by the ability to acetylate the secondary amine of acompound. In some aspects of the invention, the compound is anherbicide, e.g., glyphosate, as illustrated schematically in FIG. 1. Thecompound can also be a glyphosate analog or a metabolic product ofglyphosate degradation, e.g., aminomethylphosphonic acid. Although theacetylation of glyphosate is a key catalytic step in one metabolicpathway for catabolism of glyphosate, the enzymatic acetylation ofglyphosate by naturally-occurring, isolated, or recombinant enzymes hasnot been previously described. Thus, the nucleic acids and polypeptidesof the invention provide a new biochemical pathway for engineeringherbicide resistance.

[0050] In one aspect, the invention provides novel genes encoding GATpolypeptides. Isolated and recombinant GAT polynucleotides correspondingto naturally occurring polynucleotides, as well as recombinant andengineered, e.g., diversified, GAT polynucleotides are a feature of theinvention. GAT polynucleotides are exemplified by SEQ ID NO: 1-5,11-262, 516-567, 620, 622, 624, 626, 628, 630, 632, 634, 636, 638, 640,642, 644, 646, 648, 650, 652, 654, 656, 658, 660, 662, 664, 666, 668,670, 672, 674, 676, 678, 680, 682, 684, 686, 688, 690, 692, 694, 696,698, 700, 702, 704, 706, 708, 710, 712, 714, 716, 718, 720, 722, 724,726, 728, 730, 732, 734, 736, 738, 740, 742, 744, 746, 748, 750, 752,754, 756, 758, 760, 762, 764, 766, 768, 770, 772, 774, 776, 778, 780,782, 784, 786, 788, 790, 792, 794, 796, 798, 800, 802, 804, 806, 808,810, and 812. Specific GAT polynucleotide and polypeptide sequences areprovided as examples to help illustrate the invention, and are notintended to limit the scope of the genus of GAT polynucleotides andpolypeptides described and/or claimed herein.

[0051] The invention also provides methods for generating and selectingdiversified libraries to produce additional GAT polynucleotides,including polynucleotides encoding GAT polypeptides with improved and/orenhanced characteristics, e.g., altered K_(m) for glyphosate, increasedrate of catalysis, increased stability, etc., based upon selection of apolynucleotide constituent of the library for the new or improvedactivities described herein. Such polynucleotides are especiallyfavorably employed in the production of glyphosate resistant transgenicplants.

[0052] The GAT polypeptides of the invention exhibit a novel enzymaticactivity. Specifically, the enzymatic acetylation of the syntheticherbicide glyphosate has not been recognized prior to the presentinvention. Thus, the polypeptides herein described, e.g., as exemplifiedby SEQ ID NO: 6-10, 263-514, 568-619, 621, 623, 625, 627, 629, 631, 633,635, 637, 639, 641, 643, 645, 647, 649, 651, 653, 655, 657, 659, 661,663, 665, 667, 669, 671, 673, 675, 677, 679, 681, 683, 685, 687, 689,691, 693, 695, 697, 699, 701, 703, 705, 707, 709, 711, 713, 715, 717,719, 721, 723,725,727,729,731, 733,735,737,739, 741, 743, 745, 747, 749,751, 753, 755, 757, 759, 761, 763, 765, 767, 769, 771, 773, 775, 777,779,781,783,785,787, 789,791,793,795,797, 799,801,803,805,807, 809,811,and 813 define a novel biochemical pathway for the detoxification ofglyphosate that is functional in vivo, e.g., in plants.

[0053] Accordingly, the nucleic acids and polypeptides of the inventionare of significant utility in the generation of glyphosate resistantplants by providing new nucleic acids, polypeptides and biochemicalpathways for the engineering of herbicide selectivity in transgenicplants.

[0054] DEFINITIONS

[0055] Before describing the present invention in detail, it is to beunderstood that this invention is not limited to particular compositionsor biological systems, which can, of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting. As used in this specification and the appended claims, thesingular forms “a”, “an” and “the” include plural referents unless thecontent clearly dictates otherwise. Thus, for example, reference to “adevice” includes a combination of two or more such devices, reference to“a gene fusion construct” includes mixtures of constructs, and the like.

[0056] Unless defined otherwise, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which the invention pertains. Although any methodsand materials similar or equivalent to those described herein can beused in the practice for testing of the present invention, specificexamples of appropriate materials and methods are described herein.

[0057] In describing and claiming the present invention, the followingterminology will be used in accordance with the definitions set outbelow.

[0058] Accordingly, for purposes of the present invention, the term“glyphosate” should be considered to include any herbicidally effectiveform of N-phosphonomethylglycine (including any salt thereof) and otherforms which result in the production of the glyphosate anion in planta.The term “glyphosate analog” refers to any structural analog ofglyphosate that has the ability to inhibit EPSPS at levels such that theglyphosate analog is herbicidally effective.

[0059] As used herein, the term “glyphosate-N-acetyltransferaseactivity” or “GAT activity” refers to the ability to catalyze theacetylation of the secondary amine group of glyphosate, as illustrated,for example, in FIG. 1. A “glyphosate-N-acetyltransferase” or “GAT” isan enzyme that catalyzes the acetylation of the amine group ofglyphosate, a glyphosate analog, and/or a glyphosate primary metabolite(i.e., AMPA or sarcosine). In some preferred embodiments of theinvention, a GAT is able to transfer the acetyl group from Acetyl CoA tothe secondary amine of glyphosate and the primary amine of AMPA. Inaddition, some GATs are also able to transfer the propionyl group ofpropionyl CoA to glyphosate, indicating that GAT is also an acyltransferase. The exemplary GATs described herein are active from pH 5-9,with optimal activity in the range of pH 6.5-8.0. Activity can bequantified using various kinetic parameters which are well known in theart, e.g., K_(cat), K_(M), and k_(cat)/K_(M). These kinetic parameterscan be determined as described below in Example 7.

[0060] The terms “polynucleotide,” “nucleotide sequence,” and “nucleicacid” are used to refer to a polymer of nucleotides (A, C, T, U, G, etc.or naturally occurring or artificial nucleotide analogues), e.g., DNA orRNA, or a representation thereof, e.g., a character string, etc.,depending on the relevant context. A given polynucleotide orcomplementary polynucleotide can be determined from any specifiednucleotide sequence.

[0061] Similarly, an “amino acid sequence” is a polymer of amino acids(a protein, polypeptide, etc.) or a character string representing anamino acid polymer, depending on context. The terms “protein,”“polypeptide,” and “peptide” are used interchangeably herein.

[0062] A polynucleotide, polypeptide or other component is “isolated”when it is partially or completely separated from components with whichit is normally associated (other proteins, nucleic acids, cells,synthetic reagents, etc.). A nucleic acid or polypeptide is“recombinant” when it is artificial or engineered, or derived from anartificial or engineered protein or nucleic acid. For example, apolynucleotide that is inserted into a vector or any other heterologouslocation, e.g., in a genome of a recombinant organism, such that it isnot associated with nucleotide sequences that normally flank thepolynucleotide as it is found in nature is a recombinant polynucleotide.A protein expressed in vitro or in vivo from a recombinantpolynucleotide is an example of a recombinant polypeptide. Likewise, apolynucleotide sequence that does not appear in nature, for example avariant of a naturally occurring gene, is recombinant.

[0063] The terms “glyphosate-N-acetyl transferase polypeptide” and “GATpolypeptide” are used interchangeably to refer to any of a family ofnovel polypeptides provided herein.

[0064] The terms “glyphosate-N-acetyl transferase polynucleotide” and“GAT polynucleotide” are used interchangeably to refer to apolynucleotide that encodes a GAT polypeptide.

[0065] A “subsequence” or “fragment” is any portion of an entiresequence.

[0066] Numbering of an amino acid or nucleotide polymer corresponds tonumbering of a selected amino acid polymer or nucleic acid when theposition of a given monomer component (amino acid residue, incorporatednucleotide, etc.) of the polymer corresponds to the same residueposition in a selected reference polypeptide or polynucleotide.

[0067] A vector is a composition for facilitating celltransduction/transformation by a selected nucleic acid, or expression ofthe nucleic acid in the cell. Vectors include, e.g., plasmids, cosmids,viruses, YACs, bacteria, poly-lysine, chromosome integration vectors,episomal vectors, etc.

[0068] “Substantially an entire length of a polynucleotide or amino acidsequence” refers to at least about 70%, generally at least about 80%, ortypically about 90% or more of a sequence.

[0069] As used herein, an “antibody” refers to a protein comprising oneor more polypeptides substantially or partially encoded byimmunoglobulin genes or fragments of immunoglobulin genes. Therecognized immunoglobulin genes include the kappa, lambda, alpha, gamma,delta, epsilon and mu constant region genes, as well as myriadimmunoglobulin variable region genes. Light chains are classified aseither kappa or lambda. Heavy chains are classified as gamma, mu, alpha,delta, or epsilon, which in turn define the immunoglobulin classes, IgG,IgM, IgA, IgD and IgE, respectively. A typical immunoglobulin (antibody)structural unit comprises a tetramer. Each tetramer is composed of twoidentical pairs of polypeptide chains, each pair having one “light”(about 25 kD) and one “heavy” chain (about 50-70 kD). The N-terminus ofeach chain defines a variable region of about 100 to 110 or more aminoacids primarily responsible for antigen recognition. The terms variablelight chain (VL) and variable heavy chain (VH) refer to these light andheavy chains respectively. Antibodies exist as intact immunoglobulins oras a number of well characterized fragments produced by digestion withvarious peptidases. Thus, for example, pepsin digests an antibody belowthe disulfide linkages in the hinge region to produce F(ab)′2, a dimerof Fab which itself is a light chain joined to VH-CH1 by a disulfidebond. The F(ab)′2 may be reduced under mild conditions to break thedisulfide linkage in the hinge region thereby converting the (Fab')2dimer into an Fab' monomer. The Fab' monomer is essentially an Fab withpart of the hinge region (see, Fundamental Immunology, 4^(th) Edition,W. E. Paul (ed.), Raven Press, N.Y. (1998), for a more detaileddescription of other antibody fragments). While various antibodyfragments are defined in terms of the digestion of an intact antibody,one of skill will appreciate that such Fab′ fragments may be synthesizedde novo either chemically or by utilizing recombinant DNA methodology.Thus, the term antibody, as used herein also includes antibody fragmentseither produced by the modification of whole antibodies or synthesizedde novo using recombinant DNA methodologies. Antibodies include singlechain antibodies, including single chain Fv (sFv) antibodies in which avariable heavy and a variable light chain are joined together (directlyor through a peptide linker) to form a continuous polypeptide.

[0070] A “chloroplast transit peptide” is an amino acid sequence whichis translated in conjunction with a protein and directs the protein tothe chloroplast or other plastid types present in the cell in which theprotein is made. “Chloroplast transit sequence” refers to a nucleotidesequence that encodes a chloroplast transit peptide.

[0071] A “signal peptide” is an amino acid sequence which is translatedin conjunction with a protein and directs the protein to the secretorysystem (Chrispeels, J. J., (1991) Ann. Rev. Plant Phys. Plant Mol. Biol.42:21-53). If the protein is to be directed to a vacuole, a vacuolartargeting signal can further be added, or if to the endoplasmicreticulum, an endoplasmic reticulum retention signal may be added. Ifthe protein is to be directed to the nucleus, any signal peptide presentshould be removed and instead a nuclear localization signal included(Raikhel, N. (1992) Plant Phys. 100:1627-1632).

[0072] The terms “diversification” and “diversity,” as applied to apolynucleotide, refers to generation of a plurality of modified forms ofa parental polynucleotide, or plurality of parental polynucleotides. Inthe case where the polynucleotide encodes a polypeptide, diversity inthe nucleotide sequence of the polynucleotide can result in diversity inthe corresponding encoded polypeptide, e.g. a diverse pool ofpolynucleotides encoding a plurality of polypeptide variants. In someembodiments of the invention, this sequence diversity is exploited byscreening/selecting a library of diversified polynucleotides forvariants with desirable functional attributes, e.g., a polynucleotideencoding a GAT polypeptide with enhanced functional characteristics.

[0073] The term “encoding” refers to the ability of a nucleotidesequence to code for one or more amino acids. The term does not requirea start or stop codon. An amino acid sequence can be encoded in any oneof six different reading frames provided by a polynucleotide sequenceand its complement.

[0074] When used herein, the term “artificial variant” refers to apolypeptide having GAT activity, which is encoded by a modified GATpolynucleotide, e.g., a modified form of any one of SEQ ID NO: 1-5,11-262 516-567, 620, 622, 624, 626, 628, 630, 632, 634, 636, 638, 640,642, 644, 646, 648, 650, 652, 654, 656, 658, 660, 662, 664, 666, 668,670, 672, 674, 676, 678, 680, 682, 684, 686, 688, 690, 692, 694, 696,698, 700,702,704,706,708,710,712,714,716,718,720,722,724,726,728,730,732,734,736,738, 740, 742, 744, 746, 748, 750, 752, 754, 756, 758, 760, 762, 764,766, 768, 770, 772, 774, 776, 778, 780, 782, 784, 786, 788, 790, 792,794, 796, 798, 800, 802, 804, 806, 808, 810, and 812 or of a naturallyoccurring GAT polynucleotide isolated from an organism. The modifiedpolynucleotide, from which an artificial variant is produced whenexpressed in a suitable host, is obtained through human intervention bymodification of a GAT polynucleotide.

[0075] The term “nucleic acid construct” or “polynucleotide construct”means a nucleic acid molecule, either single- or double-stranded, whichis isolated from a naturally occurring gene or which has been modifiedto contain segments of nucleic acids in a manner that would nototherwise exist in nature. The term nucleic acid construct is synonymouswith the term “expression cassette” when the nucleic acid constructcontains the control sequences required for expression of a codingsequence of the present invention.

[0076] The term “control sequences” is defined herein to include allcomponents, which are necessary or advantageous for the expression of apolypeptide of the present invention. Each control sequence may benative or foreign to the nucleotide sequence encoding the polypeptide.Such control sequences include, but are not limited to, a leadersequence, polyadenylation sequence, propeptide sequence, promotersequence, signal peptide sequence, and transcription terminatorsequence. At a minimum, the control sequences include a promoter, andtranscriptional and translational stop signals. The control sequencesmay be provided with linkers for the purpose of introducing specificrestriction sites facilitating ligation of the control sequences withthe coding region of the nucleotide sequence encoding a polypeptide.

[0077] The term “operably linked” is defined herein as a configurationin which a control sequence is appropriately placed at a positionrelative to the coding sequence of the DNA sequence such that thecontrol sequence directs the expression of a polypeptide.

[0078] When used herein the term “coding sequence” is intended to covera nucleotide sequence, which directly specifies the amino acid sequenceof its protein product. The boundaries of the coding sequence aregenerally determined by an open reading frame, which usually begins withthe ATG start codon. The coding sequence typically includes a DNA, cDNA,and/or recombinant nucleotide sequence.

[0079] In the present context, the term “expression” includes any stepinvolved in the production of the polypeptide including, but not limitedto, transcription, post-transcriptional modification, translation,post-translational modification, and secretion.

[0080] In the present context, the term “expression vector” covers a DNAmolecule, linear or circular, that comprises a segment encoding apolypeptide of the invention, and which is operably linked to additionalsegments that provide for its transcription.

[0081] The term “host cell”, as used herein, includes any cell typewhich is susceptible to transformation with a nucleic acid construct.

[0082] The term “plant” includes whole plants, shoot vegetativeorgans/structures (e.g. leaves, stems and tubers), roots, flowers andfloral organs/structures (e.g. bracts, sepals, petals, stamens, carpels,anthers and ovules), seed (including embryo, endosperm, and seed coat)and fruit (the mature ovary), plant tissue (e.g. vascular tissue, groundtissue, and the like) and cells (e.g. guard cells, egg cells, trichomesand the like), and progeny of same. The class of plants that can be usedin the method of the invention is generally as broad as the class ofhigher and lower plants amenable to transformation techniques, includingangiosperms (monocotyledonous and dicotyledonous plants), gymnosperms,ferns, and multicellular algae. It includes plants of a variety ofploidy levels, including aneuploid, polyploid, diploid, haploid andhemizygous.

[0083] The term “heterologous” as used herein describes a relationshipbetween two or more elements which indicates that the elements are notnormally found in proximity to one another in nature. Thus, for example,a polynucleotide sequence is “heterologous to” an organism or a secondpolynucleotide sequence if it originates from a foreign species, or, iffrom the same species, is modified from its original form. For example,a promoter operably linked to a heterologous coding sequence refers to acoding sequence from a species different from that from which thepromoter was derived, or, if from the same species, a coding sequencewhich is not naturally associated with the promoter (e.g. a geneticallyengineered coding sequence or an allele from a different ecotype orvariety). An example of a heterologous polypeptide is a polypeptideexpressed from a recombinant polynucleotide in a transgenic organism.Heterologous polynucleotides and polypeptides are forms of recombinantmolecules.

[0084] A variety of additional terms are defined or otherwisecharacterized herein.

[0085] Glyphosate-N-Acetyltransferases

[0086] In one aspect, the invention provides a novel family of isolatedor recombinant enzymes referred to herein as“glyphosate-N-acetyltransferases,” “GATs,” or “GAT enzymes.” GATs areenzymes that have GAT activity, preferably sufficient activity to confersome degree of glyphosate tolerance upon a transgenic plant engineeredto express the GAT. Some examples of GATs include GAT polypeptides,described in more detail below.

[0087] GAT-mediated glyphosate tolerance is a complex function of GATactivity, GAT expression levels in the transgenic plant, the particularplant, and numerous other factors, including but not limited to, thenature and timing of herbicide application. One of skill in the art candetermine without undue experimentation the level of GAT activityrequired to effect glyphosate tolerance in a particular context.

[0088] GAT activity can be characterized using the conventional kineticparameters k_(cat), K_(M), and k_(cat) /K_(M). k_(cat) can be thought ofas a measure of the rate of acetylation, particularly at high substrateconcentrations, K_(M) is a measure of the affinity of the GAT for itssubstrates (e.g., acetyl CoA, propionyl CoA and glyphosate), andk_(cat)/K_(M) is a measure of catalytic efficiency that takes bothsubstrate affinity and catalytic rate into account. k_(cat)/K_(m) isparticularly important in the situation where the concentration of asubstrate is at least partially rate limiting. In general, a GAT with ahigher k_(cat) or k_(cat)/K_(M) is a more efficient catalyst thananother GAT with lower k_(cat) or k_(cat)/K_(M). A GAT with a lowerK_(M) is a more efficient catalyst than another GAT with a higher K_(M).Thus, to determine whether one GAT is more effective than another, onecan compare kinetic parameters for the two enzymes. The relativeimportance of k_(cat), k_(cat)/K_(M) and K_(M) will vary depending uponthe context in which the GAT will be expected to function, e.g., theanticipated effective concentration of glyphosate relative to the K_(M)for glyphosate. GAT activity can also be characterized in terms of anyof a number of functional characteristics, including, but not limitedto, stability, susceptibility to inhibition or activation by othermolecules.

[0089] Glyphosate-N-Acetyltransferase Polypeptides

[0090] In one aspect, the invention provides a novel family of isolatedor recombinant polypeptides referred to herein as“glyphosate-N-acetyltransferase polypeptides” or “GAT polypeptides.” GATpolypeptides are characterized by their structural similarity to a novelfamily of GATs. Many but not all GAT polypeptides are GATs. Thedistinction is that GATs are defined in terms of function, whereas GATpolypeptides are defined in terms of structure. A subset of the GATpolypeptides consists of those GAT polypeptides that have GAT activity,preferably at a level that will function to confer glyphosate resistanceupon a transgenic plant expressing the protein at an effective level.Some preferred GAT polypeptides for use in conferring glyphosatetolerance have a k_(cat) of at least 1 min⁻¹, or more preferably atleast 10 min⁻¹, 100 min⁻¹ or 1000 min⁻¹. Other preferred GATpolypeptides for use in conferring glyphosate tolerance have a K_(M) nogreater than 100 mM, or more preferably no greater than 10 mM, 1 mM, or0.1 mM. Still other preferred GAT polypeptides for use in conferringglyphosate tolerance have a k_(cat)/K_(M) of at least 1 mM⁻¹ min⁻¹ ormore, preferably at least 10 mM⁻¹ min⁻¹, 100 mM⁻¹ min⁻¹, 1000 mM⁻¹min⁻¹, or 10,000 mM⁻¹ min⁻¹.

[0091] Exemplary GAT polypeptides have been isolated and characterizedfrom a variety of bacterial strains. One example of a monomeric GATpolypeptide that has been isolated and characterized has a molecularradius of approximately 17 kD. An exemplary GAT enzyme isolated from astrain of B. licheniformis, SEQ ID NO:7, exhibits a K_(m) for glyphosateof approximately 2.9 mM and a K_(m) for acetyl CoA of approximately 2μM, with a k_(cat) equal to 6/minute.

[0092] The term “GAT polypeptide” refers to any polypeptide comprisingan amino acid sequence that can be optimally aligned with an amino acidsequence selected from the group consisting of SEQ ID NO: 6-10, 263-514,568-619, 621, 623, 625, 627, 629, 631, 633, 635, 637, 639, 641, 643,645, 647, 649, 651, 653, 655, 657, 659, 661, 663, 665, 667, 669, 671,673, 675, 677, 679, 681, 683, 685, 687, 689, 691, 693, 695, 697, 699,701, 703,705, 707, 709, 711, 713, 715, 717, 719, 721, 723, 725, 727,729, 731, 733, 735, 737, 739, 741, 743, 745, 747, 749, 751, 753, 755,757, 759, 761, 763, 765, 767, 769, 771, 773, 775, 777, 779, 781, 783,785, 787, 789, 791, 793, 795, 797, 799, 801, 803, 805, 807, 809, 811,and 813 to generate a similarity score of at least 460 using theBLOSUM62 matrix, a gap existence penalty of 11, and a gap extensionpenalty of 1. Some aspects of the invention pertain to GAT polypeptidescomprising an amino acid sequence that can be optimally aligned with anamino acid sequence selected from the group consisting of SEQ ID NO:6-10, 263-514, 568-619, 621, 623, 625, 627, 629, 631, 633, 635, 637,639, 641, 643, 645, 647, 649, 651, 653, 655, 657, 659, 661, 663, 665,667, 669, 671, 673, 675, 677, 679, 681, 683, 685, 687, 689, 691, 693,695, 697, 699, 701, 703, 705, 707, 709, 711, 713, 715, 717, 719, 721,723, 725, 727, 729, 731, 733, 735, 737, 739, 741, 743, 745, 747, 749,751, 753, 755, 757, 759, 761, 763, 765, 767, 769, 771, 773, 775, 777,779, 781, 783, 785, 787, 789, 791, 793, 795, 797, 799, 801, 803, 805,807, 809, 811, and 813 to generate a similarity score of at least 440,445, 450, 455, 460, 465, 470, 475, 480, 485, 490, 495, 500, 505, 510,515, 520, 525, 530, 535, 540, 545, 550, 555, 560, 565, 570, 575, 580,585, 590, 595, 600, 605, 610, 615, 620, 625, 630, 635, 640, 645, 650,655, 660, 665, 670, 675, 680, 685, 690, 695, 700, 705, 710, 715, 720,725, 730, 735, 740, 745, 750, 755, or 760 using the BLOSUM62 matrix, agap existence penalty of 11, and a gap extension penalty of 1.

[0093] One aspect of the invention pertains to a GAT polypeptidecomprising an amino acid sequence that can be optimally aligned with SEQID NO: 457 to generate a similarity score of at least 460 using theBLOSUM62 matrix, a gap existence penalty of 11, and a gap extensionpenalty of 1. Some aspects of the invention pertain to GAT polypeptidescomprising an amino acid sequence that can be optimally aligned with SEQID NO: 457 to generate a similarity score of at least 440, 445, 450,455, 460, 465, 470, 475, 480, 485, 490, 495, 500, 505, 510, 515, 520,525, 530, 535, 540, 545, 550, 555, 560, 565, 570, 575, 580, 585, 590,595, 600, 605, 610, 615, 620, 625, 630, 635, 640, 645, 650, 655, 660,665, 670, 675, 680, 685, 690, 695, 700, 705, 710, 715, 720, 725, 730,735, 740, 745, 750, 755, or 760 using the BLOSUM62 matrix, a gapexistence penalty of 11, and a gap extension penalty of 1.

[0094] One aspect of the invention pertains to a GAT polypeptidecomprising an amino acid sequence that can be optimally aligned with SEQID NO: 445 to generate a similarity score of at least 460 using theBLOSUM62 matrix, a gap existence penalty of 11, and a gap extensionpenalty of 1. Some aspects of the invention pertain to GAT polypeptidescomprising an amino acid sequence that can be optimally aligned with SEQID NO: 445 to generate a similarity score of at least 440, 445, 450,455, 460, 465, 470, 475, 480, 485, 490, 495, 500, 505, 510, 515, 520,525, 530, 535, 540, 545, 550, 555, 560, 565, 570, 575, 580, 585, 590,595, 600, 605, 610, 615, 620, 625, 630, 635, 640, 645, 650, 655, 660,665, 670, 675, 680, 685, 690, 695, 700, 705, 710, 715, 720, 725, 730,735, 740, 745, 750, 755, or 760 using the BLOSUM62 matrix, a gapexistence penalty of 11, and a gap extension penalty of 1.

[0095] One aspect of the invention pertains to a GAT polypeptidecomprising an amino acid sequence that can be optimally aligned with SEQID NO:300 to generate a similarity score of at least 460 using theBLOSUM62 matrix, a gap existence penalty of 11, and a gap extensionpenalty of 1. Some aspects of the invention pertain to GAT polypeptidescomprising an amino acid sequence that can be optimally aligned with SEQID NO: 300 to generate a similarity score of at least 440, 445, 450,455, 460, 465, 470, 475, 480, 485, 490, 495, 500, 505, 510, 515, 520,525, 530, 535, 540, 545, 550, 555, 560, 565, 570, 575, 580, 585, 590,595, 600, 605, 610, 615, 620, 625, 630, 635, 640, 645, 650, 655, 660,665, 670, 675, 680, 685, 690, 695, 700, 705, 710, 715, 720, 725, 730,735, 740, 745, 750, 755, or 760 using the BLOSUM62 matrix, a gapexistence penalty of 11, and a gap extension penalty of 1.

[0096] Two sequences are “optimally aligned” when they are aligned forsimilarity scoring using a defined amino acid substitution matrix (e.g.,BLOSUM62), gap existence penalty and gap extension penalty so as toarrive at the highest score possible for that pair of sequences. Aminoacid substitution matrices and their use in quantifying the similaritybetween two sequences are well-known in the art and described, e.g., inDayhoff et al. (1978) “A model of evolutionary change in proteins.” In“Atlas of Protein Sequence and Structure,” Vol. 5, Suppl. 3 (ed. M. O.Dayhoff), pp. 345-352. Natl. Biomed. Res. Found., Washington, D.C. andHenikoffet al. (1992) Proc. Natl. Acad. Sci. USA 89:10915-10919. TheBLOSUM62 matrix (FIG. 10) is often used as a default scoringsubstitution matrix in sequence alignment protocols such as Gapped BLAST2.0. The gap existence penalty is imposed for the introduction of asingle amino acid gap in one of the aligned sequences, and the gapextension penalty is imposed for each additional empty amino acidposition inserted into an already opened gap. The alignment is definedby the amino acids positions of each sequence at which the alignmentbegins and ends, and optionally by the insertion of a gap or multiplegaps in one or both sequences, so as to arrive at the highest possiblescore. While optimal alignment and scoring can be accomplished manually,the process is facilitated by the use of a computer-implementedalignment algorithm, e.g., gapped BLAST 2.0, described in Altschul etal, (1997) Nucleic Acids Res. 25:3389-3402, and made available to thepublic at the National Center for Biotechnology Information Website(www.ncbi.nlm.nih.gov). Optimal alignments, including multiplealignments, can be prepared using, e.g., PSI-BLAST, available throughwww.ncbi.nlm.nih.gov and described by Altschul et al, (1997) NucleicAcids Res. 25:3389-3402.

[0097] With respect to an amino acid sequence that is optimally alignedwith a reference sequence, an amino acid residue “corresponds to” theposition in the reference sequence with which the residue is paired inthe alignment. The “position” is denoted by a number that sequentiallyidentifies each amino acid in the reference sequence based on itsposition relative to the N-terminus. For example, in SEQ ID NO:300position 1 is M, position 2 is I, position 3 is E, etc. When a testsequence is optimally aligned with SEQ ID NO:300, a residue in the testsequence that aligns with the E at position 3 is said to “correspond toposition 3”of SEQ ID NO:300. Owing to deletions, insertion, truncations,fusions, etc., that must be taken into account when determining anoptimal alignment, in general the amino acid residue number in a testsequence as determined by simply counting from the N-terminal will notnecessarily be the same as the number of its corresponding position inthe reference sequence. For example, in a case where there is a deletionin an aligned test sequence, there will be no amino acid thatcorresponds to a position in the reference sequence at the site ofdeletion. Where there is an insertion in an aligned reference sequence,that insertion will not correspond to any amino acid position in thereference sequence. In the case of truncations or fusions there can bestretches of amino acids in either the reference or aligned sequencethat do not correspond to any amino acid in the corresponding sequence.

[0098] The term “GAT polypeptide” further refers to any polypeptidecomprising an amino acid sequence having at least 40% sequence identitywith an amino acid sequence selected from the group consisting of SEQ IDNO: 6-10, 263-514, 568-619, 621, 623,625,627,629,631,633,635,637,639,641, 643,645,647,649,651, 653,655,657, 659, 661, 663,665, 667, 669, 671, 673, 675, 677, 679, 681, 683, 685, 687, 689, 691,693, 695, 697, 699, 701, 703, 705, 707, 709, 711, 713, 715, 717, 719,721, 723, 725, 727, 729, 731, 733, 735, 737, 739, 741, 743, 745, 747,749, 751, 753, 755, 757, 759, 761, 763, 765, 767, 769, 771, 773, 775,777, 779, 781, 783, 785, 787, 789, 791, 793, 795, 797, 799, 801, 803,805, 807, 809, 811, and 813. Some aspects of the invention pertain toGAT polypeptides comprising an amino acid sequence having at least 60%,70%, 80%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% sequence identity with anamino acid sequence selected from the group consisting of SEQ ID NO:6-10, 263-514, 568-619, 621, 623, 625, 627, 629, 631, 633, 635, 637,639, 641, 643, 645, 647, 649, 651, 653, 655, 657, 659, 661, 663, 665,667, 669, 671, 673, 675, 677, 679, 681, 683, 685, 687, 689, 691, 693,695, 697, 699, 701, 703, 705, 707, 709, 711, 713, 715, 717, 719, 721,723, 725, 727, 729, 731, 733, 735, 737, 739, 741, 743, 745, 747, 749,751, 753, 755, 757, 759, 761, 763, 765, 767, 769, 771, 773, 775, 777,779, 781, 783, 785, 787, 789, 791, 793, 795, 797, 799, 801, 803, 805,807, 809, 811, and 813.

[0099] One aspect of the invention pertains to a GAT polypeptidecomprising an amino acid sequence having at least 40% sequence identitywith SEQ ID NO:457. Some aspects of the invention pertain to GATpolypeptides comprising an amino acid sequence having at least 60%, 70%,80%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ IDNO:457.

[0100] One aspect of the invention pertains to a GAT polypeptidecomprising an amino acid sequence having at least 40% sequence identitywith SEQ ID NO:445. Some aspects of the invention pertain to GATpolypeptides comprising an amino acid sequence having at least 60%, 70%,80%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ IDNO:445.

[0101] One aspect of the invention pertains to a GAT polypeptidecomprising an amino acid sequence having at least 40% sequence identitywith SEQ ID NO:300. Some aspects of the invention pertain to GATpolypeptides comprising an amino acid sequence having at least 60%, 70%,80%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ IDNO:300.

[0102] The term “GAT polypeptide” further refers to any polypeptidecomprising an amino acid sequence having at least 40% sequence identitywith residues 1-96 of an amino acid sequence selected from the groupconsisting of SEQ ID NO: 6-10, 263-514, 568-619, 621, 623, 625, 627,629, 631, 633, 635, 637, 639, 641, 643, 645, 647, 649, 651, 653, 655,657, 659, 661, 663, 665, 667, 669, 671, 673, 675, 677, 679, 681, 683,685, 687, 689, 691, 693, 695, 697, 699, 701, 703, 705, 707, 709, 711,713, 715, 717, 719, 721, 723, 725, 727, 729, 731, 733, 735, 737, 739,741, 743,745, 747, 749, 751, 753, 755, 757, 759, 761, 763, 765, 767,769, 771, 773, 775, 777, 779, 781, 783, 785, 787, 789, 791, 793, 795,797, 799, 801, 803, 805, 807, 809, 811, and 813. Some aspects of theinvention pertain to polypeptides comprising an amino acid sequencehaving at least 60%, 70%, 80%, 90%, 92%, 95%, 96%, 97%, 98%, or 99%sequence identity with residues 1-96 of an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 6-10, 263-514, 568-619, 621,623, 625, 627, 629, 631, 633, 635, 637, 639, 641, 643, 645, 647, 649,651, 653, 655, 657, 659, 661, 663, 665, 667, 669, 671, 673, 675, 677,679, 681, 683, 685, 687, 689, 691, 693, 695, 697, 699, 701, 703, 705,707, 709, 711, 713, 715, 717, 719, 721, 723, 725, 727, 729, 731, 733,735, 737, 739, 741, 743, 745, 747, 749, 751, 753, 755, 757, 759, 761,763, 765, 767, 769, 771, 773, 775, 777, 779, 781, 783, 785, 787, 789,791, 793, 795, 797, 799, 801, 803, 805, 807, 809, 811, and 813.

[0103] One aspect of the invention pertains to a polypeptide comprisingan amino acid sequence having at least 40% sequence identity withresidues 1-96 of SEQ ID NO:457. Some aspects of the invention pertain toGAT polypeptides comprising an amino acid sequence having at least 60%,70%, 80%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% sequence identity withresidues 1-96 of SEQ ID NO:457.

[0104] One aspect of the invention pertains to a GAT polypeptidecomprising an amino acid sequence having at least 40% sequence identitywith residues 1-96 of SEQ ID NO:445. Some aspects of the inventionpertain to GAT polypeptides comprising an amino acid sequence having atleast 60%, 70%, 80%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% sequenceidentity with residues 1-96 of SEQ ID NO:445.

[0105] One aspect of the invention pertains to a GAT polypeptidecomprising an amino acid sequence having at least 40% sequence identitywith residues 1-96 of SEQ ID NO:300. Some aspects of the inventionpertain to GAT polypeptides comprising an amino acid sequence having atleast 60%, 70%, 80%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% sequenceidentity with residues 1-96 of SEQ ID NO:300.

[0106] The term “GAT polypeptide” further refers to any polypeptidecomprising an amino acid sequence having at least 40% sequence identitywith residues 51- 146 of an amino acid sequence selected from the groupconsisting of SEQ ID NO: 6-10, 263-514, 568-619, 621, 623, 625, 627,629, 631, 633, 635, 637, 639, 641, 643, 645, 647, 649, 651, 653, 655,657, 659, 661, 663, 665, 667, 669, 671, 673, 675, 677, 679, 681, 683,685, 687, 689, 691, 693, 695, 697, 699, 701, 703, 705, 707, 709, 711,713, 715, 717, 719, 721, 723, 725, 727, 729, 731, 733, 735, 737, 739,741, 743, 745, 747, 749, 751, 753, 755, 757, 759, 761, 763, 765, 767,769, 771, 773, 775, 777, 779, 781, 783, 785, 787, 789, 791, 793, 795,797, 799, 801, 803, 805, 807, 809, 811, and 813. Some aspects of theinvention pertain to polypeptides comprising an amino acid sequencehaving at least 60%, 70%, 80%, 90%, 92%, 95%, 96%, 97%, 98%, or 99%sequence identity with residues 51-146 of an amino acid sequenceselected from the group consisting of SEQ ID NO: 6-10, 263-514, 568-619,621, 623, 625, 627, 629, 631, 633, 635, 637, 639, 641, 643, 645, 647,649, 651, 653, 655, 657, 659, 661, 663, 665, 667, 669, 671, 673, 675,677, 679, 681, 683, 685, 687, 689, 691, 693, 695, 697, 699, 701, 703,705, 707, 709, 711, 713, 715, 717, 719, 721, 723,725,727, 729, 731, 733,735, 737, 739, 741, 743, 745, 747, 749, 751, 753, 755, 757, 759, 761,763, 765, 767, 769, 771, 773, 775, 777, 779, 781, 783, 785, 787, 789,791, 793, 795, 797, 799, 801, 803, 805, 807, 809, 811, and 813.

[0107] One aspect of the invention pertains to a polypeptide comprisingan amino acid sequence having at least 40% sequence identity withresidues 51-146 of SEQ ID NO:457. Some aspects of the invention pertainto GAT polypeptides comprising an amino acid sequence having at least60%, 70%, 80%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% sequence identitywith residues 51-146 of SEQ ID NO:457.

[0108] One aspect of the invention pertains to a GAT polypeptidecomprising an amino acid sequence having at least 40% sequence identitywith residues 51-146 of SEQ ID NO:445. Some aspects of the inventionpertain to GAT polypeptides comprising an amino acid sequence having atleast 60%, 70%, 80%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% sequenceidentity with residues 51-146 of SEQ ID NO:445.

[0109] One aspect of the invention pertains to a GAT polypeptidecomprising an amino acid sequence having at least 40% sequence identitywith residues 51-146 of SEQ ID NO:300. Some aspects of the inventionpertain to GAT polypeptides comprising an amino acid sequence having atleast 60%, 70%, 80%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% sequenceidentity with residues 51-146 of SEQ ID NO:300.

[0110] As used herein, the term “identity” or “percent identity” whenused with respect to a particular pair of aligned amino acid sequences,refers to the percent amino acid sequence identity that is obtained byClustalW analysis (version W 1.8 available from European BioinfonnaticsInstitute, Cambridge, UK), counting the number of identical matches inthe alignment and dividing such number of identical matches by thegreater of (i) the length of the aligned sequences, and (ii) 96, andusing the following default ClustalW parameters to achieve slow/accuratepairwise alignments —Gap Open Penalty:10; Gap Extension Penalty:0.10;Protein weight matrix:Gonnet series; DNA weight matrix: IUB; ToggleSlow/Fast pairwise alignments =SLOW or FULL Alignment.

[0111] In another aspect, the invention provides an isolated orrecombinant polypeptide that comprises at least 20, or alternatively,50, 75, 100, 125 or 140 contiguous amino acids of an amino acid sequenceselected from the group consisting of SEQ ID NO: 6-10, 263-514, 568-619,621, 623, 625, 627, 629, 631, 633, 635, 637, 639, 641, 643, 645, 647,649, 651, 653, 655, 657, 659, 661, 663, 665, 667, 669, 671, 673, 675,677, 679, 681, 683, 685, 687, 689, 691, 693, 695, 697, 699, 701, 703,705, 707, 709, 711, 713, 715, 717, 719, 721, 723, 725, 727, 729, 731,733, 735, 737, 739, 741, 743, 745, 747, 749, 751, 753, 755, 757, 759,761, 763, 765, 767, 769, 771, 773, 775, 777, 779, 781, 783, 785, 787,789, 791, 793, 795, 797, 799, 801, 803, 805, 807, 809, 811, and 813.

[0112] In another aspect, the invention provides an isolated orrecombinant polypeptide that comprises at least 20, or alternatively,50, 75, 100, 125 or 140 contiguous amino acids of SEQ ID NO:457.

[0113] In another aspect, the invention provides an isolated orrecombinant polypeptide that comprises at least 20, or alternatively,50, 75, 100, 125 or 140 contiguous amino acids of SEQ ID NO:445.

[0114] In another aspect, the invention provides an isolated orrecombinant polypeptide that comprises at least 20, or alternatively,50, 75, 100, 125 or 140 contiguous amino acids of SEQ ID NO:300.

[0115] In another aspect, the invention provides a polypeptidecomprising an amino acid sequence selected from the group consisting ofSEQ ID NO: 6-10, 263-514, 568-619, 621, 623, 625, 627, 629, 631, 633,635, 637, 639, 641, 643, 645, 647, 649, 651, 653, 655, 657, 659, 661,663, 665, 667, 669, 671, 673, 675, 677, 679, 681, 683, 685, 687, 689,691, 693, 695, 697, 699, 701, 703, 705, 707, 709, 711, 713, 715, 717,719, 721, 723, 725, 727, 729, 731, 733, 735, 737, 739, 741, 743, 745,747, 749, 751, 753, 755, 757, 759, 761, 763, 765, 767, 769, 771, 773,775, 777, 779, 781, 783, 785, 787, 789, 791, 793, 795, 797, 799, 801,803, 805, 807, 809, 811, and 813.

[0116] Some preferred GAT polypeptides of the invention when optimallyaligned with a reference amino acid sequence selected from the groupconsisting of SEQ ID NO:6-10, 263-514, 568-619, 621, 623, 625, 627, 629,631, 633, 635, 637, 639, 641, 643, 645, 647, 649, 651, 653, 655, 657,659, 661, 663, 665, 667, 669, 671, 673, 675, 677, 679, 681, 683, 685,687, 689, 691, 693, 695, 697,699, 701, 703,705, 707,709, 711,713, 715,717, 719, 721, 723, 725, 727, 729, 731, 733, 735, 737, 739, 741, 743,745, 747, 749, 751, 753, 755, 757, 759, 761, 763, 765, 767, 769, 771,773, 775, 777, 779, 781, 783, 785, 787, 789, 791, 793, 795, 797, 799,801, 803, 805, 807, 809, 811, and 813, have at least 90% of the aminoacid residues in the polypeptide that correspond to the followingpositions conforming to the following restrictions: (a) at positions 2,4, 15, 19, 26, 28, 31, 45, 51, 54, 86, 90, 91, 97, 103, 105, 106, 114,123, 129, 139, 144, and/or 145 the amino acid residue is B1; and (b) atpositions 3, 5, 8, 10, 11, 14, 17, 18, 24, 27, 32, 37, 38, 47, 48, 49,52, 57, 58, 61, 62, 63, 68, 69, 79, 80, 82, 83, 89, 92, 100, 101, 104,119, 120, 124, 125, 126, 128, 131, 143, and/or 144 the amino acidresidue is B2; wherein B1 is an amino acid selected from the groupconsisting of A, I, L, M, F, W, Y, and V; and B2 is an amino acidselected from the group consisting of R, N, D, C, Q, E, G, H, K, P, S,and T. When used to specify an amino acid or amino acid residue, thesingle letter designations A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R,S, T, V, W, and Y have their standard meaning as used in the art and asprovided in Table 1 herein.

[0117] Some preferred GAT polypeptides of the invention when optimallyaligned with a reference amino acid sequence selected from the groupconsisting of SEQ ID NO: 6-10, 263-514, 568-619, 621, 623, 625, 627,629, 631, 633, 635, 637, 639, 641, 643, 645, 647, 649, 651, 653, 655,657, 659, 661, 663, 665, 667, 669, 671, 673, 675, 677, 679, 681, 683,685, 687, 689, 691, 693, 695, 697, 699, 701, 703, 705, 707, 709, 711,713, 715, 717, 719, 721, 723, 725, 727, 729, 731, 733, 735, 737, 739,741, 743, 745, 747, 749, 751, 753, 755, 757, 759, 761, 763, 765, 767,769, 771, 773, 775, 777, 779, 781, 783, 785, 787, 789, 791, 793, 795,797, 799, 801, 803, 805, 807, 809, 811, and 813, have at least 80% ofthe amino acid residues in the polypeptide that correspond to thefollowing positions conforming to the following restrictions: (a) atpositions 2, 4, 15, 19, 26, 28, 51, 54, 86, 90, 91, 97, 103, 105, 106,114, 129, 139, and/or 145 the amino acid residue is Z1; (b) at positions31, 45 and/or 64 the amino acid residue is Z2; (c) at positions 8, 36and/or 89 the amino acid residue is Z3 or Z6; (d) at positions 82, 92,101 and/or 120 the amino acid residue is Z4; (e) at positions 3, 11, 27and/or 79 the amino acid residue is Z5; (f) at position 123 the aminoacid residue is Z1 or Z2; (g) at positions 12, 33, 35, 39, 53, 59, 112,132, 135, 140, and/or 146 the amino acid residue is Z1 or Z3; (h) atposition 30 the amino acid residue is Z1 or Z4; (i) at position 6 theamino acid residue is Z1 or Z6; (j) at positions 81 and/or 113 the aminoacid residue is Z2 or Z3; (k) at positions 138 and/or 142 the amino acidresidue is Z2 or Z4; (1) at positions 5, 17, 24, 57, 61, 124 and/or 126the amino acid residue is Z3, Z4, or Z6; (m) at position 104 the aminoacid residue is Z3 or Z5; (o) at positions 38, 52, 62 and/or 69 theamino acid residue is Z1, Z3, Z5 or Z6; (p) at positions 14, 119 and/or144 the amino acid residue is Z1, Z2, Z4 or Z5; (q) at position 18 theamino acid residue is Z4, Z5 or Z6; (r) at positions 10, 32, 48, 63, 80and/or 83 the amino acid residue is Z5 or Z6; (s) at position 40 theamino acid residue is Z1, Z2 or Z3; (t) at positions 65 and/or 96 theamino acid residue is Z1, Z3, Z5, or Z6; (u) at positions 84 and/or 115the amino acid residue is Z1, Z3 or Z4; (v) at position 93 the aminoacid residue is Z2, Z3 or Z4; (w) at position 130 the amino acid residueis Z2, Z4 or Z6; (x) at positions 47 and/or 58 the amino acid residue isZ3, Z4 or Z6; (y) at positions 49, 68, 100 and/or 143 the amino acidresidue is Z3, Z4 or Z5; (z) at position 131 the amino acid residue isZ3, Z5 or Z6; (aa) at positions 125 and/or 128 the amino acid residue isZ4, Z5 or Z6; (ab) at position 67 the amino acid residue is Z1, Z3, Z4or Z5; (ac) at position 60 the amino acid residue is Z1, Z4, Z5 or Z6;and(ad) at position 37 the amino acid residue is Z3, Z4, Z5 or Z6;wherein Z1 is an amino acid selected from the group consisting of A, I,L, M, and V; Z2 is an amino acid selected from the group consisting ofF, W, and Y; Z3 is an amino acid selected from the group consisting ofN, Q, S, and T; Z4 is an amino acid selected from the group consistingof R, H, and K; Z5 is an amino acid selected from the group consistingof D and E; and Z6 is an amino acid selected from the group consistingof C, G, and P.

[0118] Some preferred GAT polypeptides of the invention when optimallyaligned with a reference amino acid sequence selected from the groupconsisting of SEQ ID NO: 6-10, 263-514, 568-619, 621, 623, 625, 627,629, 631, 633, 635, 637, 639, 641, 643, 645, 647, 649, 651, 653, 655,657, 659, 661, 663, 665, 667, 669, 671, 673, 675, 677, 679, 681, 683,685, 687, 689, 691, 693, 695, 697, 699, 701, 703, 705, 707, 709, 711,713, 715, 717, 719, 721, 723, 725, 727, 729, 731, 733, 735, 737, 739,741, 743, 745, 747, 749, 751, 753, 755, 757, 759, 761, 763, 765, 767,769, 771, 773, 775, 777, 779, 781, 783, 785, 787, 789, 791, 793, 795,797, 799, 801, 803, 805, 807, 809, 811, and 813, have at least 90% ofthe amino acid residues in the polypeptide that correspond to thefollowing positions conforming to the following restrictions: (a) atpositions 1, 7, 9, 13, 20, 36, 42, 46, 50, 56, 64, 70, 72, 75, 76, 78,94, 98, 107, 110, 117, 118, 121, 141 and/or 144 the amino acid residueis B1; and (b) at positions 16, 21, 22, 23, 25, 29, 34, 36, 41, 43, 44,55, 66, 71, 73, 74, 77, 85, 87, 88, 95, 99, 102, 108, 109, 111, 116,122, 127, 133, 134, 136, 137 and/or 144 the amino acid residue is B2;wherein B1 is an amino acid selected from the group consisting of A, I,L, M, F, W, Y, and V; and B2 is an amino acid selected from the groupconsisting of R, N, D, C, Q, E, G, H, K, P, S, and T.

[0119] Some preferred GAT polypeptides of the invention when optimallyaligned with a reference amino acid sequence selected from the groupconsisting of SEQ ID NO: 6-10, 263-514, 568-619, 621, 623, 625, 627,629, 631, 633, 635, 637, 639, 641, 643, 645, 647, 649, 651, 653, 655,657, 659, 661, 663, 665, 667, 669, 671, 673, 675, 677, 679, 681, 683,685, 687, 689, 691, 693, 695, 697, 699, 701, 703, 705, 707, 709, 711,713, 715, 717, 719, 721, 723, 725, 727, 729, 731, 733, 735, 737, 739,741, 743, 745, 747, 749, 751, 753, 755, 757, 759, 761, 763, 765, 767,769, 771, 773, 775, 777, 779, 781, 783, 785, 787, 789, 791, 793, 795,797, 799, 801, 803, 805, 807, 809, 811, and 813, have at least 90% ofthe amino acid residues in the polypeptide that correspond to thefollowing positions conforming to the following restrictions: (a) atpositions 1, 7, 9, 20, 36, 42, 50, 64, 72, 75, 76, 78, 94, 98, 110, 121,and/or 141 the amino acid residue is Z1; (b) at positions 13, 46, 56,64, 70, 107, 117, and/or 118 the amino acid residue is Z2; (c) atpositions 23, 36, 55, 71, 77, 88, and/or 109 the amino acid residue isZ3; (d) at positions 16, 21, 41, 73, 85, 99, and/or 111 the amino acidresidue is Z4; (e) at positions 34 and/or 95 the amino acid residue isZ5; (f) at position 22, 25, 29, 43, 44, 66, 74, 87, 102, 108, 116, 122,127, 133, 134, 136, and/or 137 the amino acid residue is Z6; wherein Z1is an amino acid selected from the group consisting of A, I, L, M, andV; Z2 is an amino acid selected from the group consisting of F, W, andY; Z3 is an amino acid selected from the group consisting of N, Q, S,and T; Z4 is an amino acid selected from the group consisting of R, H,and K; Z5 is an amino acid selected from the group consisting of D andE; and Z6 is an amino acid selected from the group consisting of C, G,and P.

[0120] In certain preferred embodiments, the GAT polypeptides of theinvention when optimally aligned with a reference amino acid sequenceselected from the group consisting of SEQ ID NO:6-10, 263-514, 568-619,621, 623, 625, 627, 629, 631, 633, 635, 637, 639, 641, 643, 645, 647,649, 651, 653, 655, 657, 659, 661, 663, 665, 667, 669, 671, 673, 675,677, 679, 681, 683, 685, 687, 689, 691, 693, 695, 697, 699, 701, 703,705, 707, 709, 711, 713, 715, 717, 719, 721, 723, 725, 727, 729, 731,733, 735, 737, 739, 741, 743, 745, 747, 749, 751, 753, 755, 757, 759,761, 763, 765, 767, 769, 771, 773, 775, 777, 779, 781, 783, 785, 787,789, 791, 793, 795, 797, 799, 801, 803, 805, 807, 809, 811, and 813,have one or more of the following positions conforming to the followingrestrictions: (a) at position 75, the amino acid is selected from thegroup consisting of B1, Z1, M or V; (b) at position 58, the amino acidis selected from the group consisting of B2, Z3, Z4, Z6, K, P, Q or R;(c) at position 47, the amino acid is selected from the group consistingof B2, Z4, Z6, R and G; (d) at position 45, the amino acid is selectedfrom the group consisting of B1, Z2, F or Y; (e) at position 91, theamino acid is selected from the group consisting of B1, Z1, L, V or I;(f) at position 105, the amino acid is selected from B1, Z1, I, M or L;(g) at position 129, the amino acid is selected from the groupconsisting of B1, Z1, I or V; and (h) at position 89, the amino acid isselected from the group consisting of B2, Z3, Z6, G, T or S.

[0121] Some preferred GAT polypeptides of the invention when optimallyaligned with a reference amino acid sequence selected from the groupconsisting of SEQ ID NO: 6-10, 263-514, 568-619, 621, 623, 625, 627,629, 631, 633, 635, 637, 639, 641, 643, 645, 647, 649, 651, 653, 655,657, 659, 661, 663, 665, 667, 669, 671, 673, 675, 677, 679, 681, 683,685, 687, 689, 691, 693, 695, 697, 699, 701, 703, 705, 707, 709, 711,713, 715, 717, 719, 721, 723, 725, 727, 729, 731, 733, 735, 737, 739,741, 743, 745, 747, 749, 751, 753, 755, 757, 759, 761, 763, 765, 767,769, 771, 773, 775, 777, 779, 781, 783, 785, 787, 789, 791, 793, 795,797, 799, 801, 803, 805, 807, 809, 811, and 813, have at least 80% ofthe amino acid residues in the polypeptide that correspond to thefollowing positions conforming to the following restrictions: (a) atposition 2 the amino acid residue is I or L; (b) at position 3 the aminoacid residue is E or D; (c) at position 4 the amino acid residue is V, Aor I; (d) at position 5 the amino acid residue is K, R or N; (e) atposition 6 the amino acid residue is P or L; (f) at position 8 the aminoacid residue is N, S or T; (g) at position 10 the amino acid residue isE or G; (h) at position 11 the amino acid residue is D or E; (i) atposition 12 the amino acid residue is T or A; (j) at position 14 theamino acid residue is D, E or K; (k) at position 15 the amino acidresidue is I or L; (l) at position 17 the amino acid residue is H or Q;(m) at position 18 the amino acid residue is E, R, C or K; (n) atposition 19 the amino acid residue is I or V; (o) at position 24 theamino acid residue is Q or R; (p) at position 26 the amino acid residueis M, V, L or I; (q) at position 27 the amino acid residue is E or D;(r) at position 28 the amino acid residue is A or V; (s) at position 30the amino acid residue is I, K, M or R; (t) at position 31 the aminoacid residue is Y or F; (u) at position 32 the amino acid residue is D,E or G; (v) at position 33 the amino acid residue is T, A or S; (w) atposition 35 the amino acid residue is L, S or M; (x) at position 37 theamino acid residue is C, R, G, E or Q; (y) at position 38 the amino acidresidue is D, G or S; (z) at position 39 the amino acid residue is T, Aor S; (aa) at position 40 the amino acid residue is F, L or S; (ab) atposition 45 the amino acid residue is Y or F; (ac) at position 47 theamino acid residue is R, Q or G; (ad) at position 48 the amino acidresidue is G or D; (ae) at position 49 the amino acid residue is K, R, Eor Q; (af) at position 51 the amino acid residue is I or V; (ag) atposition 52 the amino acid residue is S, C or G; (ah) at position 53 theamino acid residue is I, V or T; (ai) at position 54 the amino acidresidue is A or V; (aj) at position 57 the amino acid residue is H or N;(ak) at position 58 the amino acid residue is Q, K, N, R or P; (al) atposition 59 the amino acid residue is A or S; (am) at position 60 theamino acid residue is E, K, G, V or D; (an) at position 61 the aminoacid residue is H or Q; (ao) at position 62 the amino acid residue is L,P, S or T; (ap) at position 63 the amino acid residue is E, G or D; (aq)at position 65 the amino acid residue is E, D, P, V or Q; (ar) atposition 67 the amino acid residue is Q, E, R, L, H or K; (as) atposition 68 the amino acid residue is K, R, E, or N; (at) at position 69the amino acid residue is Q or P; (au) at position 79 the amino acidresidue is E or D; (av) at position 80 the amino acid residue is G or E;(aw) at position 81 the amino acid residue is H, Y, N or F; (ax) atposition 82 the amino acid residue is R or H; (ay) at position 83 theamino acid residue is E, G or D; (az) at position 84 the amino acidresidue is Q, R or L; (ba) at position 86 the amino acid residue is A orV; (bb) at position 89 the amino acid residue is G, T or S; (bc) atposition 90 the amino acid residue is L or I; (bd) at position 91 theamino acid residue is I, L or V; (be) at position 92 the amino acidresidue is R or K; (bf) at position 93 the amino acid residue is H, Y orQ; (bg) at position 96 the amino acid residue is E, A or Q; (bh) atposition 97 the amino acid residue is L or I; (bi) at position 100 theamino acid residue is K, R, N or E; (bj) at position 101 the amino acidresidue is K or R; (bk) at position 103 the amino acid residue is A orV; (bl) at position 104 the amino acid residue is D or N; (bm) atposition 105 the amino acid residue is I, L or M; (bn) at position 106the amino acid residue is L or I; (bo) at position 112 the amino acidresidue is A, T or I; (bp) at position 113 the amino acid residue is S,T or F; (bq) at position 114 the amino acid residue is A or V; (br) atposition 115 the amino acid residue is S, R or A; (bs) at position 119the amino acid residue is K, E or R; (bt) at position 120 the amino acidresidue is K or R; (bu) at position 123 the amino acid residue is F orL; (bv) at position 124 the amino acid residue is C, S or R; (bw) atposition 125 the amino acid residue is E, K, G or D; (bx) at position126 the amino acid residue is Q or H; (by) at position 128 the aminoacid residue is D, E, G or K; (bz) at position 129 the amino acidresidue is V, I or A; (ca) at position 130 the amino acid residue is Y,H, F or C; (cb) at position 131 the amino acid residue is D, G, N or E;(cc) at position 132 the amino acid residue is I, T, A, M, V or L; (cd)at position 135 the amino acid residue is V, T, A or I; (ce) at position138 the amino acid residue is H or Y; (cf) at position 139 the aminoacid residue is I or V; (cg) at position 140 the amino acid residue isL, M or S; (ch) at position 142 the amino acid residue is Y or H; (ci)at position 143 the amino acid residue is K, R, T or E; (cj) at position144 the amino acid residue is K, E, W or R; (ck) at position 145 theamino acid residue is L or I; and (cl) at position 146 the amino acidresidue is T or A.

[0122] Some preferred GAT polypeptides of the invention when optimallyaligned with a reference amino acid sequence selected from the groupconsisting of SEQ ID NO: 6-10, 263-514, 568-619, 621, 623, 625, 627,629, 631, 633, 635, 637, 639, 641, 643, 645, 647, 649, 651, 653, 655,657, 659, 661, 663, 665, 667, 669, 671, 673, 675, 677, 679, 681, 683,685, 687, 689, 691, 693, 695, 697, 699, 701, 703, 705, 707, 709, 711,713, 715, 717, 719, 721, 723, 725, 727, 729, 731, 733, 735, 737, 739,741, 743, 745, 747, 749, 751, 753, 755, 757, 759, 761, 763, 765, 767,769, 771, 773, 775, 777, 779, 781, 783, 785, 787, 789, 791, 793, 795,797, 799, 801, 803, 805, 807, 809, 811, and 813, have at least 80% ofthe amino acid residues in the polypeptide that correspond to thefollowing positions conforming to the following restrictions: (a) atposition 9, 76, 94 and 110 the amino acid residue is A; (b) at position29 and 108 the amino acid residue is C; (c) at position 34 the aminoacid residue is D; (d) at position 95 the amino acid residue is E; (e)at position 56 the amino acid residue is F; (f) at position 43, 44, 66,74, 87, 102, 116, 122, 127 and 136 the amino acid residue is G; (g) atposition 41 the amino acid residue is H; (h) at position 7 the aminoacid residue is I; (i) at position 85 the amino acid residue is K; (j)at position 20, 36, 42, 50, 72, 78, 98 and 121 the amino acid residue isL; (k) at position 1, 75 and 141 the amino acid residue is M; (l) atposition 23, 64 and 109 the amino acid residue is N; (m) at position 22,25, 133, 134 and 137 the amino acid residue is P; (n) at position 71 theamino acid residue is Q; (o) at position 16, 21, 73, 99 and 111 theamino acid residue is R; (p) at position 55 and 88 the amino acidresidue is S; (q) at position 77 the amino acid residue is T; (r) atposition 107 the amino acid residue is W; and (s) at position 13, 46,70, 117 and 118 the amino acid residue is Y.

[0123] Some preferred GAT polypeptides of the invention when optimallyaligned with a reference amino acid sequence selected from the groupconsisting of SEQ ID NO: 6-10, 263-514, and 568-619, 621, 623, 625, 627,629, 631, 633, 635, 637, 639, 641, 643, 645, 647, 649, 651, 653, 655,657, 659, 661, 663, 665, 667, 669, 671, 673, 675, 677, 679, 681, 683,685, 687, 689, 691, 693, 695, 697, 699, 701, 703, 705, 707, 709, 711,713, 715, 717, 719, 721, 723, 725, 727, 729, 731, 733, 735, 737, 739,741, 743, 745, 747, 749, 751, 753, 755, 757, 759, 761, 763, 765, 767,769, 771, 773, 775, 777, 779, 781, 783, 785, 787, 789, 791, 793, 795,797, 799, 801, 803, 805, 807, 809, 811, and 813, have the amino acidresidue in the polypeptide corresponding to position 28 is V or A.Valine or Isoleucine at the 28 position generally correlates withreduced K_(M), while alanine at that position generally correlates withincreased k_(cat). Threonine at position 89 and arginine at position 58generally correlates with reduced K_(M). Other preferred GATpolypeptides are characterized by having 127 (i.e., an I at position27), M30, D34, S35, R37, S39, G48, H41, K49, N57, Q58, P62, T62, Q65,Q67, K68, V75, E83, S89, A96, E96, R101, T 112, A 114, K119, K120, E128,V129, D131, T131, V134, V135, R144, I145, or T146, or any combinationthereof.

[0124] Some preferred GAT polypeptides of the invention comprise anamino acid sequence selected from the group consisting of SEQ IDNO:6-10, 263-514, 568-619, 621, 623, 625, 627, 629, 631, 633, 635, 637,639, 641, 643, 645, 647, 649, 651, 653, 655, 657, 659, 661, 663, 665,667, 669, 671, 673, 675, 677, 679, 681, 683, 685, 687, 689, 691, 693,695, 697, 699, 701, 703, 705, 707, 709, 711, 713, 715, 717, 719, 721,723, 725, 727, 729, 731, 733, 735, 737, 739, 741, 743, 745, 747, 749,751, 753, 755, 757, 759, 761, 763, 765, 767, 769, 771, 773, 775, 777,779, 781, 783, 785, 787, 789, 791, 793, 795, 797, 799, 801, 803, 805,807, 809, 811, and 813.

[0125] The invention further provides preferred GAT polypeptides thatare characterized by a combination of the foregoing amino acid residueposition restrictions.

[0126] In addition, the invention provides GAT polynucleotides encodingthe preferred GAT polypeptides described above, and complementarynucleotide sequences thereof.

[0127] Some aspects of the invention pertain particularly to the subsetof any of the above-described categories of GAT polypeptides having GATactivity, as described herein. These GAT polypeptides are preferred, forexample, for use as agents for conferring glyphosate resistance upon aplant. Examples of desired levels of GAT activity are described herein.

[0128] In one aspect, the GAT polypeptides comprise an amino acidsequence encoded by a recombinant or isolated form of naturallyoccurring nucleic acids isolated from a natural source, e.g., abacterial strain. Wild-type polynucleotides encoding such GATpolypeptides may be specifically screened for by standard techniquesknown in the art. The polypeptides defined by SEQ ID NO:6 to SEQ IDNO:10, for example, were discovered by expression cloning of sequencesfrom Bacillus strains exhibiting GAT activity, as described in moredetail below.

[0129] The invention also includes isolated or recombinant polypeptideswhich are encoded by an isolated or recombinant polynucleotidecomprising a nucleotide sequence which hybridizes under stringentconditions over substantially the entire length of a nucleotide sequenceselected from the group consisting of SEQ ID NO: 1-5, 11-262, 516-567,620, 622, 624, 626, 628, 630, 632, 634, 636, 638, 640, 642, 644, 646,648, 650, 652, 654, 656, 658, 660, 662, 664, 666, 668, 670, 672, 674,676, 678, 680, 682, 684, 686, 688, 690, 692, 694, 696, 698, 700, 702,704, 706, 708, 710, 712, 714, 716, 718, 720, 722, 724, 726, 728, 730,732, 734, 736, 738, 740, 742, 744, 746, 748, 750, 752, 754, 756, 758,760, 762, 764, 766, 768, 770, 772, 774, 776, 778, 780, 782, 784, 786,788, 790, 792, 794, 796, 798, 800, 802, 804, 806, 808, 810, and 812,their complements, and nucleotide sequences encoding an amino acidsequence selected from the group consisting of SEQ ID NO: 6-10, 263-514,568-619, 621, 623, 625, 627, 629, 631, 633, 635, 637, 639, 641, 643,645, 647, 649, 651, 653, 655, 657, 659, 661, 663, 665, 667, 669, 671,673, 675, 677, 679, 681, 683, 685, 687, 689, 691, 693, 695, 697, 699,701, 703, 705, 707, 709, 711, 713, 715, 717, 719, 721, 723, 725, 727,729, 731, 733, 735, 737, 739, 741, 743, 745, 747, 749, 751, 753, 755,757, 759, 761, 763, 765, 767, 769, 771, 773, 775, 777, 779, 781, 783,785, 787, 789, 791, 793, 795, 797, 799, 801, 803, 805, 807, 809, 811,and 813, including their complements.

[0130] The invention further includes any polypeptide having GATactivity that is encoded by a fragment of any of the GAT-encodingpolynucleotides described herein.

[0131] The invention also provides fragments of GAT polypeptides thatcan be spliced together to form a functional GAT polypeptide. Splicingcan be accomplished in vitro or in vivo, and can involve cis- ortrans-splicing (i.e., intramolecular or intermolecular splicing). Thefragments themselves can, but need not, have GAT activity. For example,two or more segments of a GAT polypeptide can be separated by inteins;removal of the intein sequence by cis-splicing results in a functionalGAT polypeptide. In another example, an encrypted GAT polypeptide can beexpressed as two or more separate fragments; trans-splicing of thesesegments results in recovery of a functional GAT polypeptide. Variousaspects of cis- and-trans splicing, gene encryption, and introduction ofintervening sequences are described in more detail in U.S. patentapplication Ser. Nos. 09/517,933 and 09/710,686, both of which areincorporated by reference herein in their entirety.

[0132] In general, the invention includes any polypeptide encoded by amodified GAT polynucleotide derived by mutation, recursive sequencerecombination, and/or diversification of the polynucleotide sequencesdescribed herein. In some aspects of the invention, a GAT polypeptide ismodified by single or multiple amino acid substitution, a deletion, aninsertion, or a combination of one or more of these types ofmodifications. Substitutions can be conservative, or non-conservative,can alter function or not, and can add new function. Insertions anddeletions can be substantial, such as the case of a truncation of asubstantial fragment of the sequence, or in the fusion of additionalsequence, either internally or at N or C terminal. In some embodimentsof the invention, a GAT polypeptide is part of a fusion proteincomprising a functional addition such as, for example, a secretionsignal, a chloroplast transit peptide, a purification tag, or any of thenumerous other functional groups that will be apparent to the skilledartisan, and which are described in more detail elsewhere in thisspecification.

[0133] Polypeptides of the invention may contain one or more modifiedamino acid. The presence of modified amino acids may be advantageous in,for example, (a) increasing polypeptide in vivo half-life, (b) reducingor increasing polypeptide antigenicity, and (c) increasing polypeptidestorage stability. Amino acid(s) are modified, for example,co-translationally or post-translationally during recombinant production(e.g., N-linked glycosylation at N—X—S/T motifs during expression inmammalian cells) or modified by synthetic means.

[0134] Non-limiting examples of a modified amino acid include aglycosylated amino acid, a sulfated amino acid, a prenlyated (e.g.,farnesylated, geranylgeranylated) amino acid, an acetylated amino acid,an acylated amino acid, a PEG-ylated amino acid, a biotinylated aminoacid, a carboxylated amino acid, a phosphorylated amino acid, and thelike. References adequate to guide one of skill in the modification ofamino acids are replete throughout the literature. Example protocols arefound in Walker (1998) Protein Protocols on CD-ROM Human Press, Towata,N.J.

[0135] Recombinant methods for producing and isolating GAT polypeptidesof the invention are described herein. In addition to recombinantproduction, the polypeptides may be produced by direct peptide synthesisusing solid-phase techniques (e.g., Stewart et al. (1969) Solid-PhasePeptide Synthesis, W. H. Freeman Co., San Francisco; and Merrifield J.(1963) J. Am. Chem. Soc. 85:2149-2154). Peptide synthesis may beperformed using manual techniques or by automation. Automated synthesismay be achieved, for example, using Applied Biosystems 431 A PeptideSynthesizer (Perkin Elmer, Foster City, Calif.) in accordance with theinstructions provided by the manufacturer. For example, subsequences maybe chemically synthesized separately and combined using chemical methodsto provide full-length GAT polypeptides. Peptides can also be orderedfrom a variety of sources.

[0136] In another aspect of the invention, a GAT polypeptide of theinvention is used to produce antibodies which have, e.g., diagnosticuses, for example, related to the activity, distribution, and expressionof GAT polypeptides, for example, in various tissues of a transgenicplant.

[0137] GAT homologue polypeptides for antibody induction do not requirebiological activity; however, the polypeptide or oligopeptide must beantigenic. Peptides used to induce specific antibodies may have an aminoacid sequence consisting of at least 10 amino acids, preferably at least15 or 20 amino acids. Short stretches of a GAT polypeptide may be fusedwith another protein, such as keyhole limpet hemocyanin, and antibodyproduced against the chimeric molecule.

[0138] Methods of producing polyclonal and monoclonal antibodies areknown to those of skill in the art, and many antibodies are available.See, e.g., Coligan (1991) Current Protocols in Immunology Wiley/Greene,NY; Harlow and Lane (1989) Antibodies: A Laboratory Manual Cold SpringHarbor Press, NY; Stites et al. (eds.) Basic and Clinical Immunology(4th ed.) Lange Medical Publications, Los Altos, Calif., and referencescited therein; Goding (1986) Monoclonal Antibodies: Principles andPractice (2d ed.) Academic Press, New York, N.Y.; and Kohler andMilstein (1975) Nature 256: 495-497. Other suitable techniques forantibody preparation include selection of libraries of recombinantantibodies in phage or similar vectors. See, Huse et al. (1989) Science246: 1275-1281; and Ward, et al. (1989) Nature 341: 544-546. Specificmonoclonal and polyclonal antibodies and antisera will usually bind witha K_(D) of at least about 0.1 μM, preferably at least about 0.01 μM orbetter, and most typically and preferably, 0.001 μM or better.

[0139] Additional details antibody of production and engineeringtechniques can be found in Borrebaeck (ed) (1995) Antibody Engineering,2^(nd) Edition Freeman and Company, NY (Borrebaeck); McCafferty et al.(1996) Antibody Engineering. A Practical Approach IRL at Oxford Press,Oxford, England (McCafferty), and Paul (1995) Antibody EngineeringProtocols Humana Press, Towata, N.J. (Paul).

[0140] Sequence Variations

[0141] GAT polypeptides of the present invention include conservativelymodified variations of the sequences disclosed herein as SEQ ID NO:6-10, 263-514, 568-619, 621, 623, 625, 627, 629, 631, 633, 635, 637,639, 641, 643, 645, 647, 649, 651, 653, 655, 657, 659, 661, 663, 665,667, 669, 671, 673, 675, 677, 679, 681, 683, 685, 687, 689, 691, 693,695, 697, 699, 701, 703, 705, 707, 709, 711, 713, 715, 717, 719, 721,723, 725, 727, 729, 731, 733, 735, 737, 739, 741, 743, 745, 747, 749,751, 753, 755, 757, 759, 761, 763, 765, 767, 769, 771, 773, 775, 777,779, 781, 783, 785, 787, 789, 791, 793, 795, 797, 799, 801, 803, 805,807, 809, 811, and 813. uch conservatively modified variations comprisesubstitutions, additions or deletions which alter, add or delete asingle amino acid or a small percentage of amino acids (typically lessthan about 5%, more typically less than about 4%, 2%, or 1%) in any ofSEQ ID NO: 6-10, 263-514, 568-619, 621, 623, 625, 627, 629, 631, 633,635, 637, 639, 641, 643, 645, 647, 649, 651, 653, 655, 657, 659, 661,663, 665, 667, 669, 671, 673, 675, 677, 679, 681, 683, 685, 687, 689,691, 693, 695, 697, 699, 701, 703, 705, 707, 709, 711, 713, 715, 717,719, 721, 723, 725, 727, 729, 731, 733, 735, 737,739,741,743,745,747,749,751,753,755, 757,759,761,763,765, 767,769,771, 773, 775, 777,779, 781, 783, 785, 787, 789, 791, 793, 795, 797, 799, 801, 803, 805,807, 809, 811, and 813.

[0142] For example, a conservatively modified variation (e.g., deletion)of the 146 amino acid polypeptide identified herein as SEQ ID NO:6 willhave a length of at least 140 amino acids, preferably at least 141 aminoacids, more preferably at least 144 amino acids, and still morepreferably at least 145 amino acids, corresponding to a deletion of lessthan about 5%, 4%, 2% or about 1%, or less of the polypeptide sequence.

[0143] Another example of a conservatively modified variation (e.g., a“conservatively substituted variation”) of the polypeptide identifiedherein as SEQ ID NO:6 will contain “conservative substitutions”,according to the six substitution groups set forth in Table 2, in up toabout 7 residues (i.e., less than about 5%) of the 146 amino acidpolypeptide.

[0144] The GAT polypeptide sequence homologues of the invention,including conservatively substituted sequences, can be present as partof larger polypeptide sequences such as occur in a GAT polypeptide, in aGAT fusion with a signal sequence, e.g., a chloroplast targetingsequence, or upon the addition of one or more domains for purificationof the protein (e.g., poly his segments, FLAG tag segments, etc.). Inthe latter case, the additional functional domains have little or noeffect on the activity of the GAT portion of the protein, or where theadditional domains can be removed by post synthesis processing stepssuch as by treatment with a protease.

[0145] Defining Polypeptides by Immunoreactivity

[0146] Because the polypeptides of the invention provide a new class ofenzymes with a defined activity, i.e., the acetylation and acylation ofglyphosate, the polypeptides also provide new structural features whichcan be recognized, e.g., in immunological assays. The generation ofantisera which specifically binds the polypeptides of the invention, aswell as the polypeptides which are bound by such antisera, are a featureof the invention.

[0147] The invention includes GAT polypeptides that specifically bind toor that are specifically immunoreactive with an antibody or antiseragenerated against an immunogen comprising an amino acid sequenceselected from one or more of SEQ ID NO:6-10, 263-514, 568-619, 621, 623,625, 627, 629, 631, 633, 635, 637, 639, 641, 643, 645, 647, 649, 651,653, 655, 657, 659, 661, 663, 665, 667, 669, 671, 673, 675, 677, 679,681, 683, 685, 687, 689, 691, 693, 695, 697, 699, 701, 703, 705, 707,709, 711, 713, 715, 717, 719, 721, 723, 725, 727, 729, 731, 733, 735,737, 739, 741, 743, 745, 747, 749, 751, 753, 755, 757, 759, 761, 763,765, 767, 769, 771, 773, 775, 777, 779, 781, 783, 785, 787, 789, 791,793, 795, 797, 799, 801, 803, 805, 807, 809, 811, and 813. To eliminatecross-reactivity with other GAT homologues, the antibody or antisera issubtracted with available related proteins, such as those represented bythe proteins or peptides corresponding to GenBank accession numbersavailable as of the filing date of this application, and exemplified byCAA70664, Z99109 and Y09476. Where the accession number corresponds to anucleic acid, a polypeptide encoded by the nucleic acid is generated andused for antibody/antisera subtraction purposes. FIG. 3 tabulates therelative identity between exemplary GAT sequences and the most closelyrelated sequence available in Genbank, YitI. The function of native YitIhas yet to be elucidated, but the enzyme has been shown to possessdetectable GAT activity.

[0148] In one typical format, the immunoassay uses a polyclonalantiserum which was raised against one or more polypeptides comprisingone or more of the sequences corresponding to one or more of SEQ ID NO:6-10, 263-514, 568-619, 621, 623, 625, 627, 629, 631, 633, 635, 637,639, 641, 643, 645, 647, 649, 651, 653, 655, 657, 659, 661, 663, 665,667, 669, 671, 673, 675, 677, 679, 681, 683, 685, 687, 689, 691, 693,695, 697, 699, 701, 703, 705, 707, 709, 711, 713, 715, 717, 719, 721,723, 725, 727, 729, 731, 733, 735, 737, 739, 741, 743, 745, 747, 749,751, 753, 755, 757, 759, 761, 763, 765, 767, 769, 771, 773, 775, 777,779, 781, 783, 785, 787, 789, 791, 793, 795, 797, 799, 801, 803, 805,807, 809, 811, and 813, or a substantial subsequence thereof (i.e., atleast about 30% of the full length sequence provided). The full set ofpotential polypeptide immunogens derived from SEQ ID NO: 6-10, 263-514,568-619, 621, 623, 625, 627, 629, 631, 633, 635, 637, 639, 641, 643,645, 647, 649, 651, 653, 655, 657, 659, 661, 663, 665, 667, 669, 671,673, 675, 677, 679, 681, 683, 685, 687, 689, 691, 693, 695, 697, 699,701, 703, 705, 707, 709, 711, 713, 715, 717, 719, 721, 723, 725, 727,729, 731, 733, 735, 737, 739, 741, 743, 745, 747, 749, 751, 753, 755,757, 759, 761, 763, 765, 767, 769, 771, 773, 775, 777, 779, 781, 783,785, 787, 789, 791, 793, 795, 797, 799, 801, 803, 805, 807, 809, 811,and 813 are collectively referred to below as “the immunogenicpolypeptide(s).” The resulting antisera is optionally selected to havelow cross-reactivity against other related sequences and any suchcross-reactivity is removed by immunoabsorbtion with one or more of therelated sequences, prior to use of the polyclonal antiserum in theimmunoassay.

[0149] In order to produce antisera for use in an immunoassay, one ormore of the immunogenic polypeptide(s) is produced and purified asdescribed herein. For example, recombinant protein may be produced in abacterial cell line. An inbred strain of mice (used in this assaybecause results are more reproducible due to the virtual geneticidentity of the mice) is immunized with the immunogenic polypeptide(s)in combination with a standard adjuvant, such as Freund's adjuvant,using a standard mouse immunization protocol (see, Harlow and Lane(1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Publications,New York, for a standard description of antibody generation, immunoassayformats and conditions that can be used to determine specificimmunoreactivity). Alternatively, one or more synthetic or recombinantpolypeptides derived from the sequences disclosed herein is conjugatedto a carrier protein and used as an immunogen.

[0150] Polyclonal sera are collected and titered against the immunogenicpolypeptide(s) in an immunoassay, for example, a solid phase immunoassaywith one or more of the immunogenic proteins immobilized on a solidsupport. Polyclonal antisera with a titer of 10⁶ or greater areselected, pooled and subtracted with related polypeptides, e.g., thoseidentified from GENBANK as noted, to produce subtracted, pooled, titeredpolyclonal antisera.

[0151] The subtracted, pooled, titered polyclonal antisera are testedfor cross reactivity against the related polypeptides. Preferably atleast two of the immunogenic GATs are used in this determination,preferably in conjunction with at least two related polypeptides, toidentify antibodies which are specifically bound by the immunogenicpolypeptide(s).

[0152] In this comparative assay, discriminatory binding conditions aredetermined for the subtracted, titered polyclonal antisera which resultin at least about a 5-10 fold higher signal to noise ratio for bindingof the titered polyclonal antisera to the immunogenic GAT polypeptidesas compared to binding to the related polypeptides. That is, thestringency of the binding reaction is adjusted by the addition ofnon-specific competitors such as albumin or non-fat dry milk, or byadjusting salt conditions, temperature, or the like. These bindingconditions are used in subsequent assays for determining whether a testpolypeptide is specifically bound by the pooled, subtracted polyclonalantisera. In particular, test polypeptides which show at least a 2-5fold higher signal to noise ratio than the control polypeptides underdiscriminatory binding conditions, and at least about a {fraction (1/2)} signal to noise ratio as compared to the immunogenic polypeptide(s),share substantial structural similarity with the immunogenicpolypeptide(s) as compared to known GAT, and is, therefore a polypeptideof the invention.

[0153] In another example, immunoassays in the competitive bindingformat are used for the detection of a test polypeptide. For example, asnoted, cross-reacting antibodies are removed from the pooled antiseramixture by immunoabsorption with the control GAT polypeptides. Theimmunogenic polypeptide(s) are then immobilized to a solid support whichis exposed to the subtracted pooled antisera. Test proteins are added tothe assay to compete for binding to the pooled, subtracted antisera. Theability of the test protein(s) to compete for binding to the pooled,subtracted antisera as compared to the immobilized protein(s) iscompared to the ability of the immunogenic polypeptide(s) added to theassay to compete for binding (the immunogenic polypeptide(s) competeeffectively with the immobilized immunogenic polypeptide(s) for bindingto the pooled antisera). The percent cross-reactivity for the testproteins is calculated, using standard calculations.

[0154] In a parallel assay, the ability of the control proteins tocompete for binding to the pooled, subtracted antisera is optionallydetermined as compared to the ability of the immunogenic polypeptide(s)to compete for binding to the antisera. Again, the percentcross-reactivity for the control polypeptides is calculated, usingstandard calculations. Where the percent cross-reactivity is at least5-10×higher for the test polypeptides, the test polypeptides are said tospecifically bind the pooled, subtracted antisera.

[0155] In general, the immunoabsorbed and pooled antisera can be used ina competitive binding immunoassay as described herein to compare anytest polypeptide to the immunogenic polypeptide(s). In order to makethis comparison, the two polypeptides are each assayed at a wide rangeof concentrations and the amount of each polypeptide required to inhibit50% of the binding of the subtracted antisera to the immobilized proteinis determined using standard techniques. If the amount of the testpolypeptide required is less than twice the amount of the immunogenicpolypeptide(s) that is required, then the test polypeptide is said tospecifically bind to an antibody generated to the immunogenicpolypeptide(s), provided the amount is at least about 5-10× higher asfor a control polypeptide.

[0156] As a final determination of specificity, the pooled antisera isoptionally fully immunosorbed with the immunogenic polypeptide(s)(rather than the control polypeptides) until little or no binding of thesubtracted, pooled antisera to the immunogenic polypeptide(s) isdetectable. This fully immunosorbed antisera is then tested forreactivity with the test polypeptide. If little or no reactivity isobserved (i.e., no more than 2× the signal to noise ratio observed forbinding of the fully immunosorbed antisera to the immunogenicpolypeptide(s)), then the test polypeptide is specifically bound by theantisera elicited by the immunogenic polypeptide(s).

[0157] GLYPHOSATE-N-ACETYLTRANSFERASE POLYNUCLEOTIDES

[0158] In one aspect, the invention provides a novel family of isolatedor recombinant polynucleotides referred to herein as“glyphosate-N-acetyltransferase polynucleotides” or “GATpolynucleotides.” GAT polynucleotide sequences are characterized by theability to encode a GAT polypeptide. In general, the invention includesany nucleotide sequence that encodes any of the novel GAT polypeptidesdescribed herein. In some aspects of the invention, a GAT polynucleotidethat encodes a GAT polypeptide with GAT activity is preferred.

[0159] In one aspect, the GAT polynucleotides comprise recombinant orisolated forms of naturally occurring nucleic acids isolated from anorganism, e.g., a bacterial strain. Exemplary GAT polynucleotides, e.g.,SEQ ID NO: 1 to SEQ ID NO:5, were discovered by expression cloning ofsequences from Bacillus strains exhibiting GAT activity. Briefly, acollection of approximately 500 Bacillus and Pseudomonas strains werescreened for native ability to N-acetylate glyphosate. Strains weregrown in LB overnight, harvested by centrifugation, permeabilized indilute toluene, and then washed and resuspended in a reaction mixcontaining buffer, 5 mM glyphosate, and 200 μM acetyl-CoA. The cellswere incubated in the reaction mix for between 1 and 48 hours, at whichtime an equal volume of methanol was added to the reaction. The cellswere then pelleted by centrifugation and the supernatant was filteredbefore analysis by parent ion mode mass spectrometry. The product of thereaction was positively identified as N-acetylglyphosate by comparingthe mass spectrometry profile of the reaction mix to anN-acetylglyphosate standard as shown in FIG. 2. Product detection wasdependent on inclusion of both substrates (acetyl CoA and glyphosate)and was abolished by heat denaturing the bacterial cells.

[0160] Individual GAT polynucleotides were then cloned from theidentified strains by functional screening. Genomic DNA was prepared andpartially digested with Sau3A1 enzyme. Fragments of approximately 4 Kbwere cloned into an E. coli expression vector and transformed intoelectrocompetent E. coli. Individual clones exhibiting GAT activity wereidentified by mass spectrometry following a reaction as describedpreviously except that the toluene wash was replaced by permeabilizationwith PMBS. Genomic fragments were sequenced and the putative GATpolypeptide-encoding open reading frame was identified. Identity of theGAT gene was confirmed by expression of the open reading frame in E.coli and detection of high levels of N-acetylglyphosate produced fromreaction mixtures.

[0161] In another aspect of the invention, GAT polynucleotides areproduced by diversifying, e.g., recombining and/or mutating one or morenaturally occurring, isolated, or recombinant GAT polynucleotides. Asdescribed in more detail elsewhere herein, it is often possible togenerate diversified GAT polynucleotides encoding GAT polypeptides withsuperior functional attributes, e.g., increased catalytic function,increased stability, or higher expression level, than a GATpolynucleotide used as a substrate or parent in the diversificationprocess.

[0162] The polynucleotides of the invention have a variety of uses in,for example: recombinant production (i.e., expression) of the GATpolypeptides of the invention; as transgenes (e.g., to confer herbicideresistance in transgenic plants); as selectable markers fortransformation and plasmid maintenance; as immunogens; as diagnosticprobes for the presence of complementary or partially complementarynucleic acids (including for detection of natural GAT coding nucleicacids); as substrates for further diversity generation, e.g.,recombination reactions or mutation reactions to produce new and/orimproved GAT homologues, and the like.

[0163] It is important to note that certain specific, substantial andcredible utilities of GAT polynucleotides do not require that thepolynucleotide encode a polypeptide with substantial GAT activity. Forexample, GAT polynucleotides that do not encode active enzymes can bevaluable sources of parental polynucleotides for use in diversificationprocedures to arrive at GAT polynucleotide variants, or non-GATpolynucleotides, with desirable functional properties (e.g., highk_(cat) or k_(cat)/K_(m), low K_(m), high stability towards heat orother environmental factors, high transcription or translation rates,resistance to proteolytic cleavage, reducing antigenicity, etc.). Forexample, nucleotide sequences encoding protease variants with little orno detectable activity have been used as parent polynucleotides in DNAshuffling experiments to produce progeny encoding highly activeproteases (Ness et al. (1999) Nature Biotechnology 17:893-96).

[0164] Polynucleotide sequences produced by diversity generation methodsor recursive sequence recombination (“RSR”) methods (e.g., DNAshuffling) are a feature of the invention. Mutation and recombinationmethods using the nucleic acids described herein are a feature of theinvention. For example, one method of the invention includes recursivelyrecombining one or more nucleotide sequences of the invention asdescribed above and below with one or more additional nucleotides. Therecombining steps are optionally performed in vivo, ex vivo, in silicoor in vitro. This diversity generation or recursive sequencerecombination produces at least one library of recombinant modified GATpolynucleotides. Polypeptides encoded by members of this library areincluded in the invention.

[0165] Also contemplated are uses of polynucleotides, also referred toherein as oligonucleotides, typically having at least 12 bases,preferably at least 15, more preferably at least 20, 30, or 50 or morebases, which hybridize under stringent or highly stringent conditions toa GAT polynucleotide sequence. The polynucleotides may be used asprobes, primers, sense and antisense agents, and the like, according tomethods as noted herein.

[0166] In accordance with the present invention, GAT polynucleotides,including nucleotide sequences that encode GAT polypeptides, fragmentsof GAT polypeptides, related fusion proteins, or functional equivalentsthereof, are used in recombinant DNA molecules that direct theexpression of the GAT polypeptides in appropriate host cells, such asbacterial or plant cells. Due to the inherent degeneracy of the geneticcode, other nucleic acid sequences which encode substantially the sameor a functionally equivalent amino acid sequence can also be used toclone and express the GAT polynucleotides.

[0167] The invention provides GAT polynucleotides that encodetranscription and/or translation products that are subsequently splicedto ultimately produce functional GAT polypeptides. Splicing can beaccomplished in vitro or in vivo, and can involve cis-or trans-splicing.The substrate for splicing can be polynucleotides (e.g., RNAtranscripts) or polypeptides. An example of cis-splicing of apolynucleotide is where an intron inserted into a coding sequence isremoved and the two flanking exon regions are spliced to generate a GATpolypeptide encoding sequence. An example of trans splicing would bewhere a GAT polynucleotide is encrypted by separating the codingsequence into two or more fragments that can be separately transcribedand then spliced to form the full-length GAT encoding sequence. The useof a splicing enhancer sequence (which can be introduced into aconstruct of the invention) can facilitate splicing either in cis ortrans. Cis- and trans-splicing of polypeptides are described in moredetail elsewhere herein and in U.S. patent application Ser. Nos.09/517,933 and 09/710,686.

[0168] Thus, some GAT polynucleotides do not directly encode afull-length GAT polypeptide, but rather encode a fragment or fragmentsof a GAT polypeptide. These GAT polynucleotides can be used to express afunctional GAT polypeptide through a mechanism involving splicing, wheresplicing can occur at the level of polynucleotide (e.g., intron/exon)and/or polypeptide (e.g., intein/extein). This can be useful, forexample, in controlling expression of GAT activity, since functional GATpolypeptide will only be expressed if all required fragments areexpressed in an environment that permits splicing processes to generatefunctional product. In another example, introduction of one or moreinsertion sequences into a GAT polynucleotide can facilitaterecombination with a low homology polynucleotide; use of an intron orintein for the insertion sequence facilitates the removal of theintervening sequence, thereby restoring function of the encoded variant.

[0169] As will be understood by those of skill in the art, it can beadvantageous to modify a coding sequence to enhance its expression in aparticular host. The genetic code is redundant with 64 possible codons,but most organisms preferentially use a subset of these codons. Thecodons that are utilized most often in a species are called optimalcodons, and those not utilized very often are classified as rare orlow-usage codons (see, e.g., Zhang S. P. et al. (1991) Gene 105:61-72).Codons can be substituted to reflect the preferred codon usage of thehost, a process sometimes called “codon optimization” or “controllingfor species codon bias.”

[0170] Optimized coding sequences containing codons preferred by aparticular prokaryotic or eukaryotic host (see also, Murray, E. et al.(1989) Nuc. Acids Res. 17:477-508) can be prepared, for example, toincrease the rate of translation or to produce recombinant RNAtranscripts having desirable properties, such as a longer half-life, ascompared with transcripts produced from a non-optimized sequence.Translation stop codons can also be modified to reflect host preference.For example, preferred stop codons for S. cerevisiae and mammals are UAAand UGA, respectively. The preferred stop codon for monocotyledonousplants is UGA, whereas insects and E. coli prefer to use UAA as the stopcodon (Dalphin M. E. et al. (1996) Nuc. Acids Res. 24: 216-218).Methodology for optimizing a nucleotide sequence for expression in aplant is provided, for example, in U.S. Pat. No. 6,015,891, and thereferences cited therein.

[0171] One embodiment of the invention includes a GAT polynucleotidehaving optimal codons for expression in a relevant host, e.g., atransgenic plant host. This is particularly desirable when a GATpolynucleotide of bacterial origin is introduced into a transgenicplant, e.g., to confer glyphosate resistance to the plant.

[0172] The polynucleotide sequences of the present invention can beengineered in order to alter a GAT polynucleotide for a variety ofreasons, including but not limited to, alterations which modify thecloning, processing and/or expression of the gene product. For example,alterations may be introduced using techniques that are well known inthe art, e.g., site-directed mutagenesis, to insert new restrictionsites, alter glycosylation patterns, change codon preference, introducesplice sites, etc.

[0173] As described in more detail herein, the polynucleotides of theinvention include sequences which encode novel GAT polypeptides andsequences complementary to the coding sequences, and novel fragments ofcoding sequences and complements thereof. The polynucleotides can be inthe form of RNA or in the form of DNA, and include mRNA, cRNA, syntheticRNA and DNA, genomic DNA and cDNA. The polynucleotides can bedouble-stranded or single-stranded, and if single-stranded, can be thecoding strand or the non-coding (anti-sense, complementary) strand. Thepolynucleotides optionally include the coding sequence of a GATpolypeptide (i) in isolation, (ii) in combination with an additionalcoding sequence, so as to encode, e.g., a fusion protein, a pre-protein,a prepro-protein, or the like, (iii) in combination with non-codingsequences, such as introns or inteins, control elements such as apromoter, an enhancer, a terminator element, or 5′ and/or 3′untranslated regions effective for expression of the coding sequence ina suitable host, and/or (iv) in a vector or host environment in whichthe GAT polynucleotide is a heterologous gene. Sequences can also befound in combination with typical compositional formulations of nucleicacids, including in the presence of carriers, buffers, adjuvants,excipients and the like.

[0174] Polynucleotides and oligonucleotides of the invention can beprepared by standard solid-phase methods, according to known syntheticmethods. Typically, fragments of up to about 100 bases are individuallysynthesized, then joined (e.g., by enzymatic or chemical ligationmethods, or polymerase mediated methods) to form essentially any desiredcontinuous sequence. For example, polynucleotides and oligonucleotidesof the invention can be prepared by chemical synthesis using, e.g., theclassical phosphoramidite method described by Beaucage et al. (1981)Tetrahedron Letters 22:1859-69, or the method described by Matthes etal. (1984) EMBO J. 3: 801-05, e.g., as is typically practiced inautomated synthetic methods. According to the phosphoramidite method,oligonucleotides are synthesized, e.g., in an automatic DNA synthesizer,purified, annealed, ligated and cloned in appropriate vectors.

[0175] In addition, essentially any nucleic acid can be custom orderedfrom any of a variety of commercial sources, such as The MidlandCertified Reagent Company (mcrc@oligos.com), The Great American GeneCompany (www.genco.com), ExpressGen Inc. (www.expressgen.com), OperonTechnologies Inc. (Alameda, Calif.) and many others. Similarly, peptidesand antibodies can be custom ordered from any of a variety of sources,such as PeptidoGenic (pkim@ccnet.com), HTI Bio-products, Inc.(www.htibio.com), BMA Biomedicals Ltd (U.K.), Bio.Synthesis, Inc., andmany others.

[0176] Polynucleotides may also be synthesized by well-known techniquesas described in the technical literature. See, e.g., Carruthers et al.,Cold Spring Harbor Symp. Quant. Biol. 47:411-418 (1982), and Adams etal., J Am. Chem. Soc. 105:661 (1983). Double stranded DNA fragments maythen be obtained either by synthesizing the complementary strand andannealing the strands together under appropriate conditions, or byadding the complementary strand using DNA polymerase with an appropriateprimer sequence.

[0177] General texts which describe molecular biological techniquesuseful herein, including mutagenesis, include Berger and Kimmel, Guideto Molecular Cloning Techniques Methods in Enzvmology, Volume 152,Academic Press, Inc., San Diego, Calif. (“Berger”); Sambrook et al.,Molecular Cloning-A Laboratory Manual (2nd Ed.), Volumes 1-3, ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989 (“Sambrook”);and Current Protocols in Molecular Biology, F. M. Ausubel et al., eds.,Current Protocols, a joint venture between Greene Publishing Associates,Inc. and John Wiley & Sons, Inc., (supplemented through 2000)(“Ausubel”). Examples of techniques sufficient to direct persons ofskill through in vitro amplification methods, including the polymerasechain reaction (PCR), the ligase chain reaction (LCR), Qβ-replicaseamplification and other RNA polymerase mediated techniques (e.g., NASBA)are found in Berger, Sambrook, and Ausubel, as well as in Mullis et al.,(1987) U.S. Pat. No. 4,683,202; PCR Protocols A Guide to Methods andApplications (Innis et al., eds.) Academic Press Inc. San Diego, Calif.(1990); Arnheim & Levinson (Oct. 1, 1990) Chemical and Engineering News36-47; The Journal Of NIH Research (1991) 3:81-94; Kwoh et al. (1989)Proc. Natl. Acad. Sci. USA 86:1173; Guatelli et al. (1990) Proc. Natl.Acad. Sci. USA 87:1874; Lomell et al. (1989) J. Clin. Chem. 35:1826;Landegren et al., (1988) Science 241:1077-1080; Van Brunt (1990)Biotechnology 8:291-294; Wu and Wallace, (1989) Gene 4:560; Barringer etal. (1990) Gene 89:117, and Sooknanan and Malek (1995) Biotechnology13:563-564. Improved methods of cloning in vitro amplified nucleic acidsare described in Wallace et al., U.S. Pat. No. 5,426,039. Improvedmethods of amplifying large nucleic acids by PCR are summarized in Chenget al. (1994) Nature 369:684-685 and the references cited therein, inwhich PCR amplicons of up to 40 kb are generated. One of skill willappreciate that essentially any RNA can be converted into a doublestranded DNA suitable for restriction digestion, PCR expansion andsequencing using reverse transcriptase and a polymerase. See, Ausbel,Sambrook and Berger, all supra.

[0178] Preferred polynucleotides of the present invention include anisolated or recombinant polynucleotide sequence encoding an amino acidsequence that can be optimally aligned with an amino acid sequenceselected from the group consisting of SEQ ID NO: 6-10, 263-514, 568-619,621, 623, 625, 627, 629, 631, 633, 635, 637, 639, 641, 643, 645, 647,649, 651, 653, 655, 657, 659, 661, 663, 665, 667, 669, 671, 673, 675,677, 679, 681, 683, 685, 687, 689, 691, 693, 695, 697, 699, 701, 703,705, 707, 709, 711, 713, 715, 717, 719, 721, 723, 725, 727, 729, 731,733, 735, 737, 739, 741, 743, 745, 747, 749, 751, 753, 755, 757, 759,761, 763, 765, 767, 769, 771, 773, 775, 777, 779, 781, 783, 785, 787,789, 791, 793, 795, 797, 799, 801, 803, 805, 807, 809, 811, and 813 togenerate a similarity score of at least 460 using the BLOSUM62 matrix, agap existence penalty of 11, and a gap extension penalty of 1. Someaspects of the invention pertain to an isolated or recombinantpolynucleotide sequence encoding an amino acid sequence that can beoptimally aligned with an amino acid sequence selected from the groupconsisting of SEQ ID NO: 6-10, 263-514, 568-619, 621, 623, 625, 627,629, 631, 633, 635, 637, 639, 641, 643, 645, 647, 649, 651, 653, 655,657, 659, 661, 663, 665, 667, 669, 671, 673, 675, 677, 679, 681, 683,685, 687, 689, 691, 693, 695, 697, 699, 701, 703, 705, 707, 709, 711,713, 715, 717, 719, 721, 723, 725, 727, 729, 731, 733, 735, 737, 739,741, 743, 745, 747, 749, 751, 753, 755, 757, 759, 761, 763, 765, 767,769, 771, 773, 775, 777, 779, 781, 783, 785, 787, 789, 791, 793, 795,797, 799, 801, 803, 805, 807, 809, 811, and 813 to generate a similarityscore of at least 440, 445, 450, 455, 460, 465, 470, 475, 480, 485, 490,495, 500, 505, 510, 515, 520, 525, 530, 535, 540, 545, 550, 555, 560,565, 570, 575, 580, 585, 590, 595, 600, 605, 610, 615, 620, 625, 630,635, 640, 645, 650, 655, 660, 665, 670, 675, 680, 685, 690, 695, 700,705, 710, 715, 720, 725, 730, 735, 740, 745, 750, 755; or 760 using theBLOSUM62 matrix, a gap existence penalty of 11, and a gap extensionpenalty of 1.

[0179] One aspect of the invention pertains to an isolated orrecombinant polynucleotide sequence encoding an amino acid sequence thatcan be optimally aligned with SEQ ID NO: 457 to generate a similarityscore of at least 460 using the BLOSUM62 matrix, a gap existence penaltyof 11, and a gap extension penalty of 1.Some aspects of the inventionpertain to an isolated or recombinant polynucleotide sequence encodingan amino acid sequence that can be optimally aligned with SEQ ID NO: 457to generate a similarity score of at least 440, 445, 450, 455, 460, 465,470, 475, 480, 485, 490, 495, 500, 505, 510, 515, 520, 525, 530, 535,540, 545, 550, 555, 560, 565, 570, 575, 580, 585, 590, 595, 600, 605,610, 615, 620, 625, 630, 635, 640, 645, 650, 655, 660, 665, 670, 675,680, 685, 690, 695, 700, 705, 710, 715, 720, 725, 730, 735, 740, 745,750, 755, or 760 using the BLOSUM62 matrix, a gap existence penalty of11, and a gap extension penalty of 1.

[0180] One aspect of the invention pertains to an isolated orrecombinant polynucleotide sequence encoding an amino acid sequence thatcan be optimally aligned with SEQ ID NO: 445 to generate a similarityscore of at least 460 using the BLOSUM62 matrix, a gap existence penaltyof 11, and a gap extension penalty of 1. Some aspects of the inventionpertain to an isolated or recombinant polynucleotide sequence encodingan amino acid sequence that can be optimally aligned with SEQ ID NO: 445to generate a similarity score of at least 440, 445, 450, 455, 460, 465,470, 475, 480, 485, 490, 495, 500, 505, 510, 515, 520, 525, 530, 535,540, 545, 550, 555, 560, 565, 570, 575, 580, 585, 590, 595, 600, 605,610, 615, 620, 625, 630, 635, 640, 645, 650, 655, 660, 665, 670, 675,680, 685, 690, 695, 700, 705, 710, 715, 720, 725, 730, 735, 740, 745,750, 755, or 760 using the BLOSUM62 matrix, a gap existence penalty of11, and a gap extension penalty of 1.

[0181] One aspect of the invention pertains to an isolated orrecombinant polunucleotide sequence encoding an amino acid sequence thatcan be optimally aligned with SEQ ID NO:300 to generate a similarityscore of at least 460 using the BLOSUM62 matrix, a gap existence penaltyof 11, and a gap extension penalty of 1.Some aspects of the inventionpertain to an isolated or recombinant polunucleotide sequence encodingan amino acid sequence that can be optimally aligned with SEQ ID NO: 300to generate a similarity score of at least 440, 445, 450, 455, 460, 465,470, 475, 480, 485, 490, 495, 500, 505, 510, 515, 520, 525, 530, 535,540, 545, 550, 555, 560, 565, 570, 575, 580, 585, 590, 595, 600, 605,610, 615, 620, 625, 630, 635, 640, 645, 650, 655, 660, 665, 670, 675,680, 685, 690, 695, 700, 705, 710, 715, 720, 725, 730, 735, 740, 745,750, 755, or 760 using the BLOSUM62 matrix, a gap existence penalty of11, and a gap extension penalty of 1.

[0182] The present invention further provides an isolated or recombinantpolynucleotide sequence encoding an amino acid sequence having at least40% sequence identity with an amino acid sequence selected from thegroup consisting of SEQ ID NO: 6-10, 263-514, 568-619, 621, 623, 625,627, 629, 631, 633, 635, 637, 639, 641, 643, 645, 647, 649, 651, 653,655, 657, 659, 661, 663, 665, 667, 669, 671, 673, 675, 677, 679, 681,683, 685, 687, 689, 691, 693, 695, 697, 699, 701, 703, 705, 707, 709,711, 713, 715, 717, 719, 721, 723, 725, 727, 729, 731, 733, 735, 737,739, 741, 743, 745, 747, 749, 751, 753, 755, 757, 759, 761, 763, 765,767, 769, 771, 773, 775, 777, 779, 781, 783, 785, 787, 789, 791, 793,795, 797, 799, 801, 803, 805, 807, 809, 811, and 813. Some aspects ofthe invention pertain to an isolated or recombinant polynucleotidesequence encoding an amino acid sequence having at least 60%, 70%, 80%,90%, 92%, 95%, 96%, 97%, 98%, or 99% sequence identity with an aminoacid sequence selected from the group consisting of SEQ ID NO: 6-10,263-514, 568-619, 621, 623, 625, 627, 629, 631, 633, 635, 637, 639, 641,643, 645, 647, 649, 651, 653, 655, 657, 659, 661, 663, 665, 667, 669,671, 673, 675, 677, 679, 681, 683, 685, 687, 689, 691, 693, 695, 697,699, 701, 703, 705, 707, 709, 711, 713, 715, 717, 719, 721, 723, 725,727, 729, 731, 733, 735, 737, 739, 741, 743, 745, 747, 749, 751, 753,755, 757, 759, 761, 763, 765, 767, 769, 771, 773, 775, 777, 779, 781,783, 785, 787, 789, 791, 793, 795, 797, 799, 801, 803, 805, 807, 809,811, and 813.

[0183] One aspect of the invention pertains to an isolated orrecombinant polynucleotide sequence encoding an amino acid sequencehaving at least 40% sequence identity with SEQ ID NO:457. Some aspectsof the invention pertain to an isolated or recombinant polynucleotidesequence encoding an amino acid sequence having at least 60%, 70%, 80%,90%, 92%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ IDNO:457.

[0184] One aspect of the invention pertains to an isolated orrecombinant polynucleotide sequence encoding an amino acid sequencehaving at least 40% sequence identity with SEQ ID NO:445. Some aspectsof the invention pertain to an isolated or recombinant polynucleotidesequence encoding an amino acid sequence having at least 60%, 70%, 80%,90%, 92%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ IDNO:445.

[0185] One aspect of the invention pertains to an isolated orrecombinant polynucleotide sequence encoding an amino acid sequencehaving at least 40% sequence identity with SEQ ID NO:300. Some aspectsof the invention pertain to an isolated or recombinant polynucleotidesequence encoding an amino acid sequence having at least 60%, 70%, 80%,90%, 92%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ IDNO:300.

[0186] The invention further provides an isolated or recombinantpolynucleotide sequence encoding an amino acid sequence having at least40% sequence identity with residues 1-96 of an amino acid sequenceselected from the group consisting of SEQ ID NO: 6-10, 263-514, 568-619,621, 623, 625, 627, 629, 631, 633, 635, 637, 639, 641, 643, 645, 647,649, 651, 653, 655, 657, 659, 661, 663, 665, 667, 669, 671, 673, 675,677, 679, 681, 683, 685, 687, 689, 691, 693, 695, 697, 699, 701, 703,705, 707, 709, 711, 713, 715, 717, 719, 721, 723, 725, 727, 729, 731,733, 735, 737, 739, 741, 743, 745, 747, 749, 751, 753, 755, 757, 759,761, 763, 765, 767, 769, 771, 773, 775, 777, 779, 781, 783, 785, 787,789, 791, 793, 795, 797, 799, 801, 803, 805, 807, 809, 811, and 813.Some aspects of the invention pertain to an isolated or recombinantpolynucleotide sequence encoding an amino acid sequence having at least60%, 70%, 80%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% sequence identitywith residues 1-96 of an amino acid sequence selected from the groupconsisting of SEQ ID NO: 6-10, 263-514, 568-619, 621, 623, 625, 627,629, 631, 633, 635, 637, 639, 641, 643, 645, 647, 649, 651, 653, 655,657, 659, 661, 663, 665, 667, 669, 671, 673, 675, 677, 679, 681, 683,685, 687, 689, 691, 693, 695, 697, 699, 701, 703, 705, 707, 709, 711,713, 715, 717, 719, 721, 723, 725, 727, 729, 731, 733, 735, 737, 739,741, 743, 745, 747, 749, 751, 753, 755, 757, 759, 761, 763, 765, 767,769, 771, 773, 775, 777, 779, 781, 783, 785, 787, 789, 791, 793, 795,797, 799, 801, 803, 805, 807, 809, 811, and 813.

[0187] One aspect of the invention pertains to an isolated orrecombinant polynucleotide sequence encoding an amino acid sequencehaving at least 40% sequence identity with residues 1-96 of SEQ IDNO:457. Some aspects of the invention pertain to an isolated orrecombinant polynucleotide sequence encoding an amino acid sequencehaving at least 60%, 70%, 80%, 90%, 92%, 95%, 96%, 97%, 98%, or 99%sequence identity with residues 1-96 of SEQ ID NO:457.

[0188] One aspect of the invention pertains to an isolated orrecombinant polynucleotide sequence encoding an amino acid sequencehaving at least 40% sequence identity with residues 1-96 of SEQ IDNO:445. Some aspects of the invention pertain to an isolated orrecombinant polynucleotide sequence encoding an amino acid sequencehaving at least 60%, 70%, 80%, 90%, 92%, 95%, 96%, 97%, 98%, or 99%sequence identity with residues 1-96 of SEQ ID NO:445.

[0189] One aspect of the invention pertains to an isolated orrecombinant polynucleotide sequence encoding an amino acid sequencehaving at least 40% sequence identity with residues 1-96 of SEQ IDNO:300. Some aspects of the invention pertain to an isolated orrecombinant polynucleotide sequence encoding an amino acid sequencehaving at least 60%, 70%, 80%, 90%, 92%, 95%, 96%, 97%, 98%, or 99%sequence identity with residues 1-96 of SEQ ID NO:300.

[0190] The invention further provides an isolated or recombinantpolynucleotide sequence encoding an amino acid sequence having at least40% sequence identity with residues 51-146 of an amino acid sequenceselected from the group consisting of SEQ ID NO: 6-10, 263-514, 568-619,621, 623, 625, 627, 629, 631, 633, 635, 637, 639, 641, 643, 645, 647,649, 651, 653, 655, 657, 659, 661, 663, 665, 667, 669, 671, 673, 675,677, 679, 681, 683, 685, 687, 689, 691, 693, 695, 697, 699, 701, 703,705, 707, 709, 711, 713, 715, 717, 719, 721, 723, 725, 727, 729, 731,733, 735, 737, 739, 741, 743, 745, 747, 749, 751, 753, 755, 757, 759,761, 763, 765, 767, 769, 771, 773, 775, 777, 779, 781, 783, 785, 787,789, 791, 793, 795, 797, 799, 801, 803, 805, 807, 809, 811, and 813.Some aspects of the invention pertain to an isolated or recombinantpolynucleotide sequence encoding an amino acid sequence having at least60%, 70%, 80%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% sequence identitywith residues 51-146 of an amino acid sequence selected from the groupconsisting of SEQ ID NO: 6-10, 263-514, 568-619, 621, 623, 625, 627,629, 631, 633, 635, 637, 639, 641, 643, 645, 647, 649, 651, 653, 655,657, 659, 661, 663, 665, 667, 669, 671, 673, 675, 677, 679, 681, 683,685, 687, 689, 691, 693, 695, 697, 699, 701, 703, 705, 707, 709, 711,713, 715, 717, 719, 721, 723, 725, 727, 729, 731, 733, 735, 737, 739,741, 743, 745, 747, 749, 751, 753, 755, 757, 759, 761, 763, 765, 767,769, 771, 773, 775, 777, 779, 781, 783, 785, 787, 789, 791, 793, 795,797, 799, 801, 803, 805, 807, 809, 811, and 813.

[0191] One aspect of the invention pertains to an isolated orrecombinant polynucleotide sequence encoding an amino acid sequencehaving at least 40% sequence identity with residues 51- 146 of SEQ IDNO:457. Some aspects of the invention pertain to an isolated orrecombinant polynucleotide sequence encoding an amino acid sequencehaving at least 60%, 70%, 80%, 90%, 92%, 95%, 96%, 97%, 98%, or 99%sequence identity with residues 51-146 of SEQ ID NO:457.

[0192] One aspect of the invention pertains to an isolated orrecombinant polynucleotide sequence encoding an amino acid sequencehaving at least 40% sequence identity with residues 51-146 of SEQ IDNO:445. Some aspects of the invention pertain to an isolated orrecombinant polynucleotide sequence encoding an amino acid sequencehaving at least 60%, 70%, 80%, 90%, 92%, 95%, 96%, 97%, 98%, or 99%sequence identity with residues 51-146 of SEQ ID NO:445.

[0193] One aspect of the invention pertains to an isolated orrecombinant polynucleotide sequence encoding an amino acid sequencehaving at least 40% sequence identity with residues 51-146 of SEQ IDNO:300. Some aspects of the invention pertain to an isolated orrecombinant polynucleotide sequence encoding an amino acid sequencehaving at least 60%, 70%, 80%, 90%, 92%, 95%, 96%, 97%, 98%, or 99%sequence identity with residues 51-146 of SEQ ID NO:300.

[0194] Further, an isolated or recombinant polynucleotide sequenceencoding an amino acid that comprises at least 20, or alternatively, 50,75, 100, 125 or 140 contiguous amino acids of an amino acid sequenceselected from the group consisting of SEQ ID NO: 6-10, 263-514, 568-619,621, 623, 625, 627, 629, 631, 633, 635, 637, 639, 641, 643, 645, 647,649, 651, 653, 655, 657, 659, 661, 663, 665, 667, 669, 671, 673, 675,677, 679, 681, 683, 685, 687, 689, 691, 693, 695, 697, 699, 701, 703,705, 707, 709, 711, 713, 715, 717, 719, 721, 723, 725, 727, 729, 731,733, 735, 737, 739, 741, 743, 745, 747, 749, 751, 753, 755, 757, 759,761, 763, 765, 767, 769, 771, 773, 775, 777, 779, 781, 783, 785, 787,789, 791, 793, 795, 797, 799, 801, 803, 805, 807, 809, 811, and 813 isprovided.

[0195] In another aspect, the invention provides an isolated orrecombinant polynucleotide encoding an amino acid that comprises atleast 20, or alternatively, 50, 75, 100, 125 or 140 contiguous aminoacids of SEQ ID NO:457.

[0196] In another aspect, the invention provides an isolated orrecombinant polynucleotide encoding an amino acid that comprises atleast 20, or alternatively, 50, 75, 100, 125 or 140 contiguous aminoacids of SEQ ID NO:445.

[0197] In another aspect, the invention provides an isolated orrecombinant polynucleotide encoding an amino acid that comprises atleast 20, or alternatively, 50, 75, 100, 125 or 140 contiguous aminoacids of SEQ ID NO:300.

[0198] In another aspect, the invention provides an isolated orrecombinant polynucleotide sequence encoding an amino acid sequenceselected from the group consisting of SEQ ID NO: 6-10, 263-514, 568-619,621, 623, 625, 627, 629, 631, 633, 635, 637, 639, 641, 643, 645, 647,649, 651, 653, 655, 657, 659, 661, 663, 665, 667, 669, 671, 673, 675,677, 679, 681, 683, 685, 687, 689, 691, 693, 695, 697, 699, 701, 703,705, 707, 709, 711, 713, 715, 717, 719, 721, 723, 725, 727, 729, 731,733, 735, 737, 739, 741, 743, 745, 747, 749, 751, 753, 755, 757, 759,761, 763, 765, 767, 769, 771, 773, 775, 777, 779, 781, 783, 785, 787,789, 791, 793, 795, 797, 799, 801, 803, 805, 807, 809, 811, and 813.

[0199] Some preferred isolated or recombinant polynucleotides of theinvention encode an amino acid sequence that when optimally aligned witha reference amino acid sequence selected from the group consisting ofSEQ ID NO:6-10, 263-514, 568-619, 621, 623, 625, 627, 629, 631, 633,635, 637, 639, 641, 643, 645, 647, 649, 651, 653, 655, 657, 659, 661,663, 665, 667, 669, 671, 673, 675, 677, 679, 681, 683, 685, 687, 689,691, 693, 695, 697, 699, 701, 703, 705, 707, 709, 711, 713, 715, 717,719, 721, 723, 725, 727, 729, 731, 733, 735, 737, 739, 741, 743, 745,747, 749, 751, 753, 755, 757, 759, 761, 763, 765, 767, 769, 771, 773,775, 777, 779, 781, 783, 785, 787, 789, 791, 793, 795, 797, 799, 801,803, 805, 807, 809, 811, and 813, have at least 90% of the amino acidresidues in the polypeptide that correspond to the following positionsconforming to the following restrictions: (a) at positions 2, 4, 15, 19,26, 28, 31, 45, 51, 54, 86, 90, 91, 97, 103, 105, 106, 114, 123, 129,139, 144, and/or 145 the amino acid residue is B1; and (b) at positions3, 5, 8, 10, 11, 14, 17, 18, 24, 27, 32, 37, 38, 47,48, 49, 52, 57, 58,61, 62, 63, 68, 69, 79, 80, 82, 83, 89, 92, 100, 101, 104, 119, 120,124, 125, 126, 128, 131, 143, and/or 144 the amino acid residue is B2;wherein B1 is an amino acid selected from the group consisting of A, I,L, M, F, W, Y, and V; and B2 is an amino acid selected from the groupconsisting of R, N, D, C, Q, E, G, H, K, P, S, and T. When used tospecify an amino acid or amino acid residue, the single letterdesignations A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W,and Y have their standard meaning as used in the art and as provided inTable 1 herein.

[0200] Some preferred isolated or recombinant polynucleotides of theinvention encode an amino acid sequence that when optimally aligned witha reference amino acid sequence selected from the group consisting ofSEQ ID NO:6-10, 263-514, 568-619, 621, 623, 625, 627, 629, 631, 633,635, 637, 639, 641, 643, 645, 647, 649, 651, 653, 655, 657, 659, 661,663, 665, 667, 669, 671, 673, 675, 677, 679, 681, 683, 685, 687, 689,691, 693, 695, 697, 699, 701, 703, 705, 707, 709, 711, 713, 715, 717,719, 721, 723, 725, 727, 729, 731, 733, 735, 737, 739, 741, 743, 745,747, 749, 751, 753, 755, 757, 759, 761, 763, 765, 767, 769, 771, 773,775, 777, 779, 781, 783, 785, 787, 789, 791, 793, 795, 797, 799, 801,803, 805, 807, 809, 811, and 813, have at least 80% of the amino acidresidues in the polypeptide that correspond to the following positionsconforming to the following restrictions: (a) at positions 2,4, 15, 19,26, 28, 51, 54, 86, 90, 91, 97, 103, 105, 106, 114, 129, 139, and/or 145the amino acid residue is Z1; (b) at positions 31, 45 and/or 64 theamino acid residue is Z2; (c) at positions 8, 36 and/or 89 the aminoacid residue is Z3 or Z6; (d) at positions 82, 92, 101 and/or 120 theamino acid residue is Z4; (e) at positions 3, 11, 27 and/or 79 the aminoacid residue is Z5; (f) at position 123 the amino acid residue is Z1 orZ2; (g) at positions 12, 33, 35, 39, 53, 59, 112, 132, 135, 140, and/or146 the amino acid residue is Z1 or Z3; (h) at position 30 the aminoacid residue is Z1 or Z4; (i) at position 6 the amino acid residue is Z1or Z6; (j) at positions 81 and/or 113 the amino acid residue is Z2 orZ3; (k) at positions 138 and/or 142 the amino acid residue is Z2 or Z4;(1) at positions 5, 17, 24, 57, 61, 124 and/or 126 the amino acidresidue is Z3, Z4, or Z6; (m) at position 104 the amino acid residue isZ3 or Z5; (o) at positions 38, 52, 62 and/or 69 the amino acid residueis Z1, Z3, Z5 or Z6; (p) at positions 14, 119 and/or 144 the amino acidresidue is Z1, Z2, Z4 or Z5; (q) at position 18 the amino acid residueis Z4, Z5 or Z6; (r) at positions 10, 32, 48, 63, 80 and/or 83 the aminoacid residue is Z5 or Z6; (s) at position 40 the amino acid residue isZ1, Z2 or Z3; (t) at positions 65 and/or 96 the amino acid residue isZ1, Z3, Z5, or Z6; (u) at positions 84 and/or 115 the amino acid residueis Z1, Z3 or Z4; (v) at position 93 the amino acid residue is Z2, Z3 orZ4; (w) at position 130 the amino acid residue is Z2, Z4 or Z6; (x) atpositions 47 and/or 58 the amino acid residue is Z3, Z4 or Z6; (y) atpositions 49, 68, 100 and/or 143 the amino acid residue is Z3, Z4 or Z5;(z) at position 131 the amino acid residue is Z3, Z5 or Z6; (aa) atpositions 125 and/or 128 the amino acid residue is Z4, Z5 or Z6; (ab) atposition 67 the amino acid residue is Z1, Z3, Z4 or Z5; (ac) at position60 the amino acid residue is Z1, Z4, Z5 or Z6; and(ad) at position 37the amino acid residue is Z3, Z4, Z5 or Z6; wherein Z1 is an amino acidselected from the group consisting of A, I, L, M, and V; Z2 is an aminoacid selected from the group consisting of F, W, and Y; Z3 is an aminoacid selected from the group consisting of N, Q, S, and T; Z4 is anamino acid selected from the group consisting of R, H, and K; Z5 is anamino acid selected from the group consisting of D and E; and Z6 is anamino acid selected from the group consisting of C, G, and P.

[0201] Some preferred isolated or recombinant polynucleotides of theinvention encode an amino acid sequence that when optimally aligned witha reference amino acid sequence selected from the group consisting ofSEQ ID NO:6-10, 263-514, 568-619, 621, 623, 625, 627, 629, 631, 633,635, 637, 639, 641, 643, 645, 647, 649, 651, 653, 655, 657, 659, 661,663, 665, 667, 669, 671, 673, 675, 677, 679, 681, 683, 685, 687, 689,691, 693, 695, 697, 699, 701, 703, 705, 707, 709, 711, 713, 715, 717,719, 721, 723, 725, 727, 729, 731, 733, 735, 737, 739, 741, 743, 745,747, 749, 751, 753, 755, 757, 759, 761, 763, 765, 767, 769, 771, 773,775, 777, 779, 781, 783, 785, 787, 789, 791, 793, 795, 797, 799, 801,803, 805, 807, 809, 811, and 813, have at least 90% of the amino acidresidues in the polypeptide that correspond to the following positionsconforming to the following restrictions: (a) at positions 1, 7, 9, 13,20, 36, 42, 46, 50, 56, 64, 70, 72, 75, 76, 78, 94, 98, 107, 110, 117,118, 121, 141 and/or 144 the amino acid residue is B1; and (b) atpositions 16, 21, 22, 23, 25, 29, 34, 36, 41, 43, 44, 55, 66, 71, 73,74, 77, 85, 87, 88, 95, 99, 102, 108, 109, 111, 116, 122, 127, 133, 134,136, 137 and/or 144 the amino acid residue is B2; wherein B1 is an aminoacid selected from the group consisting of A, I, L, M, F, W, Y, and V;and B2 is an amino acid selected from the group consisting of R, N, D,C, Q, E, G, H, K, P, S, and T.

[0202] Some preferred isolated or recombinant polynucleotides of theinvention encode an amino acid sequence that when optimally aligned witha reference amino acid sequence selected from the group consisting ofSEQ ID NO:6-10, 263-514, 568-619, 621, 623, 625, 627, 629, 631, 633,635, 637, 639, 641, 643, 645, 647, 649, 651, 653, 655, 657, 659, 661,663, 665, 667, 669, 671, 673, 675, 677, 679, 681, 683, 685, 687, 689,691, 693, 695, 697, 699, 701, 703, 705, 707, 709, 711, 713, 715,717,719, 721, 723, 725, 727, 729, 731, 733, 735, 737, 739, 741, 743,745, 747, 749, 751, 753, 755, 757, 759, 761, 763, 765, 767, 769, 771,773, 775, 777, 779, 781, 783, 785, 787, 789, 791, 793, 795, 797, 799,801, 803, 805, 807, 809, 811, and 813, have at least 90% of the aminoacid residues in the polypeptide that correspond to the followingpositions conforming to the following restrictions: (a) at positions 1,7, 9, 20, 36, 42, 50, 64, 72, 75, 76, 78, 94, 98, 110, 121, and/or 141the amino acid residue is Z1; (b) at positions 13, 46, 56, 64, 70, 107,117, and/or 118 the amino acid residue is Z2; (c) at positions 23, 36,55, 71, 77, 88, and/or 109 the amino acid residue is Z3; (d) atpositions 16, 21, 41, 73, 85, 99, and/or 111 the amino acid residue isZ4; (e) at positions 34 and/or 95 the amino acid residue is Z5; (f) atposition 22, 25, 29, 43, 44, 66, 74, 87, 102, 108, 116, 122, 127, 133,134, 136, and/or 137 the amino acid residue is Z6; wherein Z1 is anamino acid selected from the group consisting of A, I, L, M, and V; Z2is an amino acid selected from the group consisting of F, W, and Y; Z3is an amino acid selected from the group consisting of N, Q, S, and T;Z4 is an amino acid selected from the group consisting of R, H, and K;Z5 is an amino acid selected from the group consisting of D and E; andZ6 is an amino acid selected from the group consisting of C, G, and P.

[0203] In certain preferred embodiments, the isolated or recombinantpolynucleotides of the invention encode an amino acid sequence that whenoptimally aligned with a reference amino acid sequence selected from thegroup consisting of SEQ ID NO:6-10, 263-514, 568-619, 621, 623, 625,627, 629, 631, 633, 635, 637, 639, 641, 643, 645, 647, 649, 651, 653,655, 657, 659, 661, 663, 665, 667, 669, 671, 673, 675, 677, 679, 681,683, 685, 687, 689, 691, 693, 695, 697, 699, 701, 703, 705, 707, 709,711, 713, 715, 717, 719, 721, 723, 725, 727, 729, 731, 733, 735, 737,739, 741, 743, 745, 747, 749, 751, 753, 755, 757, 759, 761, 763, 765,767, 769, 771, 773, 775, 777, 779, 781, 783, 785, 787, 789, 791, 793,795, 797, 799, 801, 803, 805, 807, 809, 811, and 813, have one or moreof the following positions conforming to the following restrictions: (a)at position 75, the amino acid is selected from the group consisting ofB1, Z1, M or V; (b) at position 58, the amino acid is selected from thegroup consisting of B2, Z3, Z4, Z6, K, P, Q or R; (c) at position 47,the amino acid is selected from the group consisting of B2, Z4, Z6, Rand G; (d) at position 45, the amino acid is selected from the groupconsisting of B 1, Z2, F or Y; (e) at position 91, the amino acid isselected from the group consisting of B1, Z1, L, V or I; (f) at position105, the amino acid is selected from B1, Z1, I, M or L; (g) at position129, the amino acid is selected from the group consisting of B 1, Z 1, Ior V; and (h) at position 89, the amino acid is selected from the groupconsisting of B2, Z3, Z6, G, T or S.

[0204] Some preferred isolated or recombinant polynucleotides of theinvention encode an amino acid sequence that when optimally aligned witha reference amino acid sequence selected from the group consisting ofSEQ ID NO:6-10, 263-514, 568-619, 621, 623, 625, 627, 629, 631, 633,635, 637, 639, 641, 643, 645, 647, 649, 651, 653, 655, 657, 659, 661,663, 665, 667, 669, 671, 673, 675, 677, 679, 681, 683, 685, 687, 689,691, 693, 695, 697, 699, 701, 703, 705, 707, 709, 711, 713, 715, 717,719, 721, 723, 725, 727, 729, 731, 733, 735, 737, 739, 741, 743, 745,747, 749, 751, 753, 755, 757, 759, 761, 763, 765, 767, 769, 771, 773,775, 777, 779, 781, 783, 785, 787, 789, 791, 793, 795, 797, 799, 801,803, 805, 807, 809, 811, and 813, have at least 80% of the amino acidresidues in the polypeptide that correspond to the following positionsconforming to the following restrictions: (a) at position 2 the aminoacid residue is I or L; (b) at position 3 the amino acid residue is E orD; (c) at position 4 the amino acid residue is V, A or I; (d) atposition 5 the amino acid residue is K, R or N; (e) at position 6 theamino acid residue is P or L; (f) at position 8 the amino acid residueis N, S or T; (g) at position 10 the amino acid residue is E or G; (h)at position 11 the amino acid residue is D or E; (i) at position 12 theamino acid residue is T or A; (j) at position 14 the amino acid residueis D, E or K; (k) at position 15 the amino acid residue is I or L; (l)at position 17 the amino acid residue is H or Q; (m) at position 18 theamino acid residue is E, R, C or K; (n) at position 19 the amino acidresidue is I or V; (o) at position 24 the amino acid residue is Q or R;(p) at position 26 the amino acid residue is M, V, L or I; (q) atposition 27 the amino acid residue is E or D; (r) at position 28 theamino acid residue is A or V; (s) at position 30 the amino acid residueis I, K, M or R; (t) at position 31 the amino acid residue is Y or F;(u) at position 32 the amino acid residue is D, E or G; (v) at position33 the amino acid residue is T, A or S; (w) at position 35 the aminoacid residue is L, S or M; (x) at position 37 the amino acid residue isC, R, G, E or Q; (y) at position 38 the amino acid residue is D, G or S;(z) at position 39 the amino acid residue is T, A or S; (aa) at position40 the amino acid residue is F, L or S; (ab) at position 45 the aminoacid residue is Y or F; (ac) at position 47 the amino acid residue is R,Q or G; (ad) at position 48 the amino acid residue is G or D; (ae) atposition 49 the amino acid residue is K, R, E or Q; (at) at position 51the amino acid residue is I or V; (ag) at position 52 the amino acidresidue is S, C or G; (ah) at position 53 the amino acid residue is I, Vor T; (ai) at position 54 the amino acid residue is A or V; (aj) atposition 57 the amino acid residue is H or N; (ak) at position 58 theamino acid residue is Q, K, N, R or P; (al) at position 59 the aminoacid residue is A or S; (am) at position 60 the amino acid residue is E,K, G, V or D; (an) at position 61 the amino acid residue is H or Q; (ao)at position 62 the amino acid residue is L, P, S or T; (ap) at position63 the amino acid residue is E, G or D; (aq) at position 65 the aminoacid residue is E, D, P, V or Q; (ar) at position 67 the amino acidresidue is Q, E, R, L, H or K; (as) at position 68 the amino acidresidue is K, R, E, or N; (at) at position 69 the amino acid residue isQ or P; (au) at position 79 the amino acid residue is E or D; (av) atposition 80 the amino acid residue is G or E; (aw) at position 81 theamino acid residue is H, Y, N or F; (ax) at position 82 the amino acidresidue is R or H; (ay) at position 83 the amino acid residue is E, G orD; (az) at position 84 the amino acid residue is Q, R or L; (ba) atposition 86 the amino acid residue is A or V; (bb) at position 89 theamino acid residue is G, T or S; (be) at position 90 the amino acidresidue is L or I; (bd) at position 91 the amino acid residue is I, L orV; (be) at position 92 the amino acid residue is R or K; (bf) atposition 93 the amino acid residue is H, Y or Q; (bg) at position 96 theamino acid residue is E, A or Q; (bh) at position 97 the amino acidresidue is L or I; (bi) at position 100 the amino acid residue is K, R,N or E; (bj) at position 101 the amino acid residue is K or R; (bk) atposition 103 the amino acid residue is A or V; (bl) at position 104 theamino acid residue is D or N; (bm) at position 105 the amino acidresidue is I, L or M; (bn) at position 106 the amino acid residue is Lor I (bo) at position 112 the amino acid residue is A, T or I; (bp) atposition 113 the amino acid residue is S, T or F; (bq) at position 114the amino acid residue is A or V; (br) at position 115 the amino acidresidue is S, R or A; (bs) at position 119 the amino acid residue is K,E or R; (bt) at position 120 the amino acid residue is K or R; (bu) atposition 123 the amino acid residue is F or L; (bv) at position 124 theamino acid residue is C, S or R; (bw) at position 125 the amino acidresidue is E, K, G or D; (bx) at position 126 the amino acid residue isQ or H; (by) at position 128 the amino acid residue is D, E, G or K;(bz) at position 129 the amino acid residue is V, I or A; (ca) atposition 130 the amino acid residue is Y, H, F or C; (cb) at position131 the amino acid residue is D, G, N or E; (cc) at position 132 theamino acid residue is I, T, A, M, V or L; (cd) at position 135 the aminoacid residue is V, T, A or I; (ce) at position 138 the amino acidresidue is H or Y; (cf) at position 139 the amino acid residue is I orV; (cg) at position 140 the amino acid residue is L, M or S; (ch) atposition 142 the amino acid residue is Y or H; (ci) at position 143 theamino acid residue is K, R, T or E; (cj) at position 144 the amino acidresidue is K, E, W or R; (ck) at position 145 the amino acid residue isL or I; and (cl) at position 146 the amino acid residue is T or A.

[0205] Some preferred isolated or recombinant polynucleotides of theinvention encode an amino acid sequence that when optimally aligned witha reference amino acid sequence selected from the group consisting ofSEQ ID NO:6-10, 263-514, 568-619, 621, 623, 625, 627, 629, 631, 633,635, 637, 639, 641, 643, 645, 647, 649, 651, 653, 655, 657, 659, 661,663, 665, 667, 669, 671, 673, 675, 677, 679, 681, 683, 685, 687, 689,691, 693,695,697,699,701,703,705,707,709,711,713,715,717,719,721,723,725,727,729,731, 733, 735, 737, 739, 741, 743, 745, 747, 749, 751, 753, 755, 757,759, 761, 763, 765, 767, 769, 771, 773, 775, 777, 779, 781, 783, 785,787, 789, 791, 793, 795, 797, 799, 801, 803, 805, 807, 809, 811, and813, have at least 80% of the amino acid residues in the polypeptidethat correspond to the following positions conforming to the followingrestrictions: (a) at position 9, 76, 94 and 110 the amino acid residueis A; (b) at position 29 and 108 the amino acid residue is C; (c) atposition 34 the amino acid residue is D; (d) at position 95 the aminoacid residue is E; (e) at position 56 the amino acid residue is F; (f)at position 43, 44, 66, 74, 87, 102, 116, 122, 127 and 136 the aminoacid residue is G; (g) at position 41 the amino acid residue is H; (h)at position 7 the amino acid residue is I; (i) at position 85 the aminoacid residue is K; (j) at position 20, 36, 42, 50, 72, 78, 98 and 121the amino acid residue is L; (k) at position 1, 75 and 141 the aminoacid residue is M; (l) at position 23, 64 and 109 the amino acid residueis N; (m) at position 22, 25, 133, 134 and 137 the amino acid residue isP; (n) at position 71 the amino acid residue is Q; (o) at position 16,21, 73, 99 and 111 the amino acid residue is R; (p) at position 55 and88 the amino acid residue is S; (q) at position 77 the amino acidresidue is T; (r) at position 107 the amino acid residue is W; and (s)at position 13, 46, 70, 117 and 118 the amino acid residue is Y.

[0206] Some preferred isolated or recombinant polynucleotides of theinvention encode an amino acid that when optimally aligned with areference amino acid sequence selected from the group consisting of SEQID NO:6-10, 263-514, and 568-619, 621, 623, 625, 627, 629, 631, 633,635, 637, 639, 641, 643, 645, 647, 649, 651, 653, 655, 657, 659, 661,663, 665, 667, 669, 671, 673, 675, 677, 679, 681, 683, 685, 687, 689,691, 693, 695, 697, 699, 701, 703, 705, 707, 709, 711, 713, 715, 717,719, 721, 723, 725, 727, 729, 731, 733, 735, 737, 739, 741, 743, 745,747, 749, 751, 753, 755, 757, 759, 761, 763, 765, 767, 769, 771, 773,775, 777, 779, 781, 783, 785, 787, 789, 791, 793, 795, 797, 799, 801,803, 805, 807, 809, 811, and 813, have the amino acid residue in thepolypeptide corresponding to position 28 is V or A. Valine or Isoleucineat the 28 position generally correlates with reduced K_(M), whilealanine at that position generally correlates with increased k_(cat).Threonine at position 89 and arginine at position 58 generallycorrelates with reduced K_(M). Other preferred GAT polypeptides arecharacterized by having 127 (i.e., an I at position 27), M30, D34, S35,R37, S39, G48, H41, K49, N57, Q58, P62, T62, Q65, Q67, K68, V75, E83,S89, A96, E96, R101, T112, A114, K119, K120, E128, V129, D131, T131,V134, V135, R144, 1145, or T146, or any combination thereof.

[0207] Some preferred isolated or recombinant polynucleotides of theinvention comprise a nucleotide sequence selected from the groupconsisting of SEQ ID NO: 1-5, 11-262,516-567, 620, 622, 624, 626, 628,630, 632, 634, 636, 638, 640, 642, 644, 646, 648, 650, 652, 654, 656,658, 660, 662, 664, 666, 668, 670, 672, 674, 676, 678, 680, 682, 684,686, 688, 690, 692, 694, 696, 698, 700, 702, 704, 706, 708, 710, 712,714, 716, 718, 720, 722, 724, 726, 728, 730, 732, 734, 736, 738, 740,742, 744, 746, 748, 750, 752, 754, 756, 758, 760, 762, 764, 766, 768,770, 772, 774, 776, 778, 780, 782, 784, 786, 788, 790, 792, 794, 796,798, 800, 802, 804, 806, 808, 810, and 812.

[0208] Sequence Variations

[0209] It will be appreciated by those skilled in the art that due tothe degeneracy of the genetic code, a multitude of nucleotide sequencesencoding GAT polypeptides of the invention may be produced, some ofwhich bear substantial identity to the nucleic acid sequences explicitlydisclosed herein. TABLE 1 Codon Table Amino acids Codon Alanine Ala AGCA GCC GCG GCU Cysteine Cys C UGG UGU Aspartic acid Asp D GAC GAUGlutamic acid Glu E GAA GAG Phenylalanine Phe F UUG UUU Glycine Gly GGGA GGC GGG GGU Histidine His H CAC CAU Isoleucine Ile I AUA AUC AUULysine Lys K AAA AAG Leucine Leu L UUA UUG CUA CUC CUG CUU MethionineMet M AUG Asparagine Asn N AAG AAU Proline Pro P CCA CCC CCG CCUGlutamine Gln Q CAA CAG Arginine Arg R AGA AGG CGA CGC CGG CGU SerineSer S AGC AGU UGA UCC UCG UCU Threonine Thr T ACA ACC ACG ACU Valine ValV GUA GUC GUG GUU Tryptophan Trp W UGG Tyrosine Tyr Y UAC UAU

[0210] For instance, inspection of the codon table (Table 1) shows thatcodons AGA, AGG, CGA, CGC, CGG, and CGU all encode the amino acidarginine. Thus, at every position in the nucleic acids of the inventionwhere an arginine is specified by a codon, the codon can be altered toany of the corresponding codons described above without altering theencoded polypeptide. It is understood that U in an RNA sequencecorresponds to T in a DNA sequence.

[0211] Using, as an example, the nucleic acid sequence corresponding tonucleotides 1-15 of SEQ ID NO:1, ATG ATT GAA GTC AAA (SEQ ID NO:826), asilent variation of this sequence includes AGT ATC GAG GTG AAG (SEQ IDNO:827), both sequences which encode the amino acid sequence MIEVK (SEQID NO:828), corresponding to amino acids 1-5 of SEQ ID NO:6.

[0212] Such “silent variations” are one species of “conservativelymodified variations”, discussed below. One of skill will recognize thateach codon in a nucleic acid (except AUG, which is ordinarily the onlycodon for methionine) can be modified by standard techniques to encode afunctionally identical polypeptide. Accordingly, each silent variationof a nucleic acid which encodes a polypeptide is implicit in anydescribed sequence. The invention provides each and every possiblevariation of nucleic acid sequence encoding a polypeptide of theinvention that could be made by selecting combinations based on possiblecodon choices. These combinations are made in accordance with thestandard triplet genetic code (e.g., as set forth in Table 1) as appliedto the nucleic acid sequence encoding a GAT homologue polypeptide of theinvention. All such variations of every nucleic acid herein arespecifically provided and described by consideration of the sequence incombination with the genetic code. Any variant can be produced as notedherein.

[0213] A group of two or more different codons that, when translated inthe same context, all encode the same amino acid, are referred to hereinas “synonymous codons.” As described herein, in some aspects of theinvention a GAT polynucleotide is engineered for optimized codon usagein a desired host organism, for example a plant host. The term“optimized” or “optimal” are not meant to be restricted to the very bestpossible combination of codons, but simple indicates that the codingsequence as a whole possesses an improved usage of codons relative to aprecursor polynucleotide from which it was derived. Thus, in one aspectthe invention provides a method for producing a GAT polynucleotidevariant by replacing at least one parental codon in a nucleotidesequence with a synonymous codon that is preferentially used in adesired host organism, e.g., a plant, relative to the parental codon.

[0214] “Conservatively modified variations” or, simply, “conservativevariations” of a particular nucleic acid sequence refers to thosenucleic acids which encode identical or essentially identical amino acidsequences, or, where the nucleic acid does not encode an amino acidsequence, to essentially identical sequences. One of skill willrecognize that individual substitutions, deletions or additions whichalter, add or delete a single amino acid or a small percentage of aminoacids (typically less than 5%, more typically less than 4%, 2% or 1%, orless) in an encoded sequence are “conservatively modified variations”where the alterations result in the deletion of an amino acid, additionof an amino acid, or substitution of an amino acid with a chemicallysimilar amino acid.

[0215] Conservative substitution tables providing functionally similaramino acids are well known in the art. Table 2 sets forth six groupswhich contain amino acids that are “conservative substitutions” for oneanother. TABLE 2 Conservative Substitution Groups 1 Alanine (A) Serine(S) Threonine (T) 2 Aspartic acid (D) Glutamic acid (E) 3 Asparagine (N)Glutamine (Q) 4 Arginine (R) Lysine (K) 5 Isoleucine (I) Leucine (L)Methionine (M) Valine (V) 6 Phenylalanine (F) Tyrosine (Y) Tryptophan(W)

[0216] Thus, “conservatively substituted variations” of a listedpolypeptide sequence of the present invention include substitutions of asmall percentage, typically less than 5%, more typically less than 2%and often less than 1%, of the amino acids of the polypeptide sequence,with a conservatively selected amino acid of the same conservativesubstitution group.

[0217] For example, a conservatively substituted variation of thepolypeptide identified herein as SEQ ID NO:6 will contain “conservativesubstitutions”, according to the six groups defined above, in up to 7residues (i.e., 5% of the amino acids) in the 146 amino acidpolypeptide.

[0218] In a further example, if four conservative substitutions werelocalized in the region corresponding to amino acids 21 to 30 of SEQ IDNO:6, examples of conservatively substituted variations of this region,

[0219] RPN QPL EAC M (SEQ ID NO:829), include:

[0220]KPQ QPV ESC M (SEQ ID NO: 830) and

[0221]KPN NPL DAC V (SEQ ID NO:831) and the like, in accordance with the

[0222] conservative substitutions listed in Table 2 (in the aboveexample, conservative substitutions are underlined). The listing of aprotein sequence herein, in conjunction with the above substitutiontable, provides an express listing of all conservatively substitutedproteins.

[0223] Finally, the addition of sequences which do not alter the encodedactivity of a nucleic acid molecule, such as the addition of anon-functional or non-coding sequence, is a conservative variation ofthe basic nucleic acid.

[0224] One of skill will appreciate that many conservative variations ofthe nucleic acid constructs which are disclosed yield a functionallyidentical construct. For example, as discussed above, owing to thedegeneracy of the genetic code, “silent substitutions” (i.e.,substitutions in a nucleic acid sequence which do not result in analteration in an encoded polypeptide) are an implied feature of everynucleic acid sequence which encodes an amino acid. Similarly,“conservative amino acid substitutions,” in one or a few amino acids inan amino acid sequence are substituted with different amino acids withhighly similar properties, are also readily identified as being highlysimilar to a disclosed construct. Such conservative variations of eachdisclosed sequence are a feature of the present invention.

[0225] Non-conservative modifications of a particular nucleic acid arethose which substitute any amino acid not characterized as aconservative substitution. For example, any substitution which crossesthe bounds of the six groups set forth in Table 2. These includesubstitutions of basic or acidic amino acids for neutral amino acids,(e.g., Asp, Glu, Asn, or Gln for Val, Ile, Leu or Met), aromatic aminoacid for basic or acidic amino acids (e.g., Phe, Tyr or Trp for Asp,Asn, Glu or Gln) or any other substitution not replacing an amino acidwith a like amino acid.

[0226] Nucleic Acid Hybridization

[0227] Nucleic acids “hybridize” when they associate, typically insolution. Nucleic acids hybridize due to a variety of well-characterizedphysico-chemical forces, such as hydrogen bonding, solvent exclusion,base stacking and the like. An extensive guide to the hybridization ofnucleic acids is found in Tijssen (1993) Laboratory Techniques inBiochemistry and Molecular Biology—Hybridization with Nucleic AcidProbes, Part I, Chapter 2, “Overview of principles of hybridization andthe strategy of nucleic acid probe assays,” (Elsevier, N.Y.(“Tijssen”)), as well as in Ausubel, supra, Hames and Higgins (1995)Gene Probes 1, IRL Press at Oxford University Press, Oxford, England(“Hames and Higgins 1”) and Hames and Higgins (1995) Gene Probes 2, IRLPress at Oxford University Press, Oxford, England (“Hames and Higgins2”) and provide details on the synthesis, labeling, detection andquantification of DNA and RNA, including oligonucleotides.

[0228] “Stringent hybridization wash conditions” in the context ofnucleic acid hybridization experiments, such as Southern and northernhybridizations, are sequence dependent, and are different underdifferent environmental parameters. An extensive guide to thehybridization of nucleic acids is found in Tijssen (1993), supra, and inHames and Higgins 1 and Hames and Higgins 2, supra.

[0229] For purposes of the present invention, generally, “highlystringent” hybridization and wash conditions are selected to be about 5°C. or less lower than the thermal melting point (T_(m)) for the specificsequence at a defined ionic strength and pH (as noted below, highlystringent conditions can also be referred to in comparative terms).

[0230] The T_(m) is the temperature (under defined ionic strength andpH) at which 50% of the test sequence hybridizes to a perfectly matchedprobe. Very stringent conditions are selected to be equal to the T_(m)for a particular probe.

[0231] The T_(m) of a nucleic acid duplex indicates the temperature atwhich the duplex is 50% denatured under the given conditions and itsrepresents a direct measure of the stability of the nucleic acid hybrid.Thus, the T_(m) corresponds to the temperature corresponding to themidpoint in transition from helix to random coil and it depends onlength, nucleotide composition, and ionic strength for long stretches ofnucleotides.

[0232] After hybridization, unhybridized nucleic acid material can beremoved by a series of washes, the stringency of which can be adjusteddepending upon the desired results. Low stringency washing conditions(e.g., using higher salt and lower temperature) increase sensitivity,but can produce nonspecific hybridization signals and high backgroundsignals. Higher stringency conditions (e.g., using lower salt and highertemperature that is closer to the hybridization temperature) lowers thebackground signal, typically with only the specific signal remaining.See Rapley, R. and Walker, J. M. eds., Molecular Biomethods Handbook(Humana Press, Inc. 1998) (hereinafter “Rapley and Walker”), which isincorporated herein by reference in its entirety for all purposes.

[0233] The T_(m) of a DNA-DNA duplex can be estimated using Equation 1as follows:

T _(m)(° C.)=81.5° C.+16.6(log₁₀ M)+0.41(%G+C)−0.72(%f)−500/n,

[0234] where M is the molarity of the monovalent cations (usually Na+),(%G+C) is the percentage of guanosine (G) and cytosine (C) nucleotides,(%f) is the percentage of formalize and n is the number of nucleotidebases (i.e., length) of the hybrid. See Rapley and Walker, supra.

[0235] The T_(m) of an RNA-DNA duplex can be estimated by using Equation2 as follows:

T _(m)(° C.)=79.8° C.+18.5(log₁₀ M)+0.58(%G+C)−11.8(%G+C)²−0.56(%f)−820/n,

[0236] where M is the molarity of the monovalent cations (usually Na+),(%G+C) is the percentage of guanosine (G ) and cytosine (C) nucleotides,(%f) is the percentage of formamide and n is the number of nucleotidebases (i.e., length) of the hybrid. Id.

[0237] Equations 1 and 2 are typically accurate only for hybrid duplexeslonger than about 100-200 nucleotides. Id.

[0238] The T_(m) of nucleic acid sequences shorter than 50 nucleotidescan be calculated as follows:

T _(m)(° C.)=4(G+C)+2(A+T),

[0239] where A (adenine), C, T (thymine), and G are the numbers of thecorresponding nucleotides.

[0240] An example of stringent hybridization conditions forhybridization of complementary nucleic acids which have more than 100complementary residues on a filter in a Southern or northern blot is 50%formalin with 1 mg of heparin at 42° C., with the hybridization beingcarried out overnight. An example of stringent wash conditions is a 0.2×SSC wash at 65° C. for 15 minutes (see Sambrook, supra for a descriptionof SSC buffer). Often the high stringency wash is preceded by a lowstringency wash to remove background probe signal. An example lowstringency wash is 2× SSC at 40° C. for 15 minutes.

[0241] In general, a signal to noise ratio of 2.5×-5× (or higher) thanthat observed for an unrelated probe in the particular hybridizationassay indicates detection of a specific hybridization. Detection of atleast stringent hybridization between two sequences in the context ofthe present invention indicates relatively strong structural similarityor homology to, e.g., the nucleic acids of the present inventionprovided in the sequence listings herein.

[0242] As noted, “highly stringent” conditions are selected to be about5° C. or less lower than the thermal melting point (T_(m)) for thespecific sequence at a defined ionic strength and pH. Target sequencesthat are closely related or identical to the nucleotide sequence ofinterest (e.g., “probes”) can be identified under highly stringentconditions. Lower stringency conditions are appropriate for sequencesthat are less complementary. See, e.g., Rapley and Walker, supra.

[0243] Comparative hybridization can be used to identify nucleic acidsof the invention, and this comparative hybridization method is apreferred method of distinguishing nucleic acids of the invention.Detection of highly stringent hybridization between two nucleotidesequences in the context of the present invention indicates relativelystrong structural similarity/homology to, e.g., the nucleic acidsprovided in the sequence listing herein. Highly stringent hybridizationbetween two nucleotide sequences demonstrates a degree of similarity orhomology of structure, nucleotide base composition, arrangement or orderthat is greater than that detected by stringent hybridizationconditions. In particular, detection of highly stringent hybridizationin the context of the present invention indicates strong structuralsimilarity or structural homology (e.g., nucleotide structure, basecomposition, arrangement or order) to, e.g., the nucleic acids providedin the sequence listings herein. For example, it is desirable toidentify test nucleic acids that hybridize to the exemplar nucleic acidsherein under stringent conditions.

[0244] Thus, one measure of stringent hybridization is the ability tohybridize to one of the listed nucleic acids (e.g., nucleic acidsequences SEQ ID NO:1-5, 11-262,516-567, 620, 622, 624, 626, 628, 630,632, 634, 636, 638, 640, 642, 644, 646, 648, 650, 652, 654, 656, 658,660, 662, 664, 666, 668, 670, 672, 674, 676, 678, 680, 682, 684, 686,688, 690,692,694,696,698, 700,702,704,706,708, 710,712,714,716,718,720,722,724, 726, 728, 730, 732, 734, 736, 738, 740, 742, 744, 746, 748,750, 752, 754, 756, 758, 760, 762, 764, 766, 768, 770, 772, 774, 776,778, 780, 782, 784, 786, 788, 790, 792, 794, 796, 798, 800, 802, 804,806, 808, 810, and 812, and complementary polynucleotide sequencesthereof), under highly stringent conditions (or very stringentconditions, or ultra-high stringency hybridization conditions, orultra-ultra high stringency hybridization conditions). Stringenthybridization (as well as highly stringent, ultra-high stringency, orultra-ultra high stringency hybridization conditions) and washconditions can easily be determined empirically for any test nucleicacid. For example, in determining highly stringent hybridization andwash conditions, the hybridization and wash conditions are graduallyincreased (e.g., by increasing temperature, decreasing saltconcentration, increasing detergent concentration and/or increasing theconcentration of organic solvents, such as formalin, in thehybridization or wash), until a selected set of criteria are met. Forexample, the hybridization and wash conditions are gradually increaseduntil a probe comprising one or more nucleic acid sequences selectedfrom SEQ ID NO:1-5, 11-262, 516-567, 620, 622, 624, 626, 628, 630, 632,634, 636, 638, 640, 642, 644, 646, 648, 650, 652, 654, 656, 658, 660,662, 664, 666, 668, 670, 672, 674, 676, 678, 680, 682, 684, 686, 688,690, 692, 694, 696, 698, 700, 702, 704, 706, 708, 710, 712, 714, 716,718, 720, 722, 724, 726, 728, 730, 732, 734, 736, 738, 740, 742, 744,746, 748, 750, 752, 754, 756, 758, 760, 762, 764, 766, 768, 770, 772,774, 776, 778, 780, 782, 784, 786, 788, 790, 792, 794, 796, 798, 800,802, 804, 806, 808, 810, and 812, and complementary polynucleotidesequences thereof, binds to a perfectly matched complementary target(again, a nucleic acid comprising one or more nucleic acid sequencesselected from SEQ ID NO: 1-5, 11-262, 516-567, 620, 622, 624, 626, 628,630, 632, 634, 636, 638, 640, 642, 644, 646, 648, 650, 652, 654, 656,658, 660, 662, 664, 666, 668, 670, 672, 674, 676, 678, 680, 682, 684,686, 688, 690, 692, 694, 696, 698, 700, 702, 704, 706, 708, 710, 712,714, 716, 718, 720, 722, 724, 726, 728, 730, 732, 734, 736, 738, 740,742, 744, 746, 748, 750, 752, 754, 756, 758, 760, 762, 764, 766, 768,770, 772, 774, 776, 778, 780, 782, 784, 786, 788, 790, 792, 794, 796,798, 800, 802, 804, 806, 808, 810, and 812, and complementarypolynucleotide sequences thereof), with a signal to noise ratio that isat least about 2.5×, and optionally about 5× or more as high as thatobserved for hybridization of the probe to an unmatched target. In thiscase, the unmatched target is a nucleic acid corresponding to a nucleicacid (other than those in the accompanying sequence listing) that ispresent in a public database such as GenBank™ at the time of filing ofthe subject application. Such sequences can be identified in GenBank byone of skill. Examples include Accession Nos. Z99109 and Y09476.Additional such sequences can be identified in e.g., GenBank, by one ofordinary skill in the art.

[0245] A test nucleic acid is said to specifically hybridize to a probenucleic acid when it hybridizes at least ½ as well to the probe as tothe perfectly matched complementary target, i.e., with a signal to noiseratio at least ½ as high as hybridization of the probe to the targetunder conditions in which the perfectly matched probe binds to theperfectly matched complementary target with a signal to noise ratio thatis at least about 2×-10×, and occasionally 20×, 50× or greater than thatobserved for hybridization to any of the unmatched polynucleotides ofAccession Nos. Z99109 and Y09476.

[0246] Ultra high-stringency hybridization and wash conditions are thosein which the stringency of hybridization and wash conditions areincreased until the signal to noise ratio for binding of the probe tothe perfectly matched complementary target nucleic acid is at least 10×as high as that observed for hybridization to any of the unmatchedtarget nucleic acids of Genbank Accession numbers Z99109 and Y09476. Atarget nucleic acid which hybridizes to a probe under such conditions,with a signal to noise ratio of at least ½ that of the perfectly matchedcomplementary target nucleic acid is said to bind to the probe underultra-high stringency conditions.

[0247] Similarly, even higher levels of stringency can be determined bygradually increasing the hybridization and/or wash conditions of therelevant hybridization assay. For example, those in which the stringencyof hybridization and wash conditions are increased until the signal tonoise ratio for binding of the probe to the perfectly matchedcomplementary target nucleic acid is at least 10×, 20×, 50×, 100×, or500× or more as high as that observed for hybridization to any of theunmatched target nucleic acids of Genbank Accession numbers Z99109 andY09476. A target nucleic acid which hybridizes to a probe under suchconditions, with a signal to noise ratio of at least ½ that of theperfectly matched complementary target nucleic acid is said to bind tothe probe under ultra-ultra-high stringency conditions.

[0248] Target nucleic acids which hybridize to the nucleic acidsrepresented by SEQ ID NO:1-5, 11-262,516-567, 620, 622, 624, 626, 628,630, 632, 634, 636, 638, 640, 642, 644, 646, 648, 650, 652, 654, 656,658, 660, 662, 664, 666, 668, 670, 672, 674, 676, 678, 680, 682, 684,686, 688, 690, 692, 694, 696, 698, 700, 702, 704, 706, 708, 710, 712,714, 716, 718, 720, 722, 724, 726, 728, 730, 732, 734, 736, 738, 740,742, 744, 746, 748, 750, 752, 754, 756, 758, 760, 762, 764, 766, 768,770, 772, 774, 776, 778, 780, 782, 784, 786, 788, 790, 792, 794, 796,798, 800, 802, 804, 806, 808, 810, and 812 under high, ultra-high andultra-ultra high stringency conditions are a feature of the invention.Examples of such nucleic acids include those with one or a few silent orconservative nucleic acid substitutions as compared to a given nucleicacid sequence.

[0249] Nucleic acids which do not hybridize to each other understringent conditions are still substantially identical if thepolypeptides which they encode are substantially identical. This occurs,e.g., when a copy of a nucleic acid is created using the maximum codondegeneracy permitted by the genetic code, or when antisera or antiserumgenerated against one or more of SEQ ID NO:6-10, 263-514,568-619, 621,623, 625, 627, 629, 631, 633, 635, 637, 639, 641, 643, 645, 647, 649,651, 653, 655, 657, 659, 661, 663, 665, 667, 669, 671, 673, 675, 677,679, 681, 683, 685, 687, 689, 691, 693, 695, 697, 699,701,703,705,707,709, 711,713,715,717,719, 721,723,725,727,729,731,733,735, 737, 739, 741, 743, 745, 747, 749, 751, 753, 755, 757, 759,761, 763, 765, 767, 769, 771, 773, 775, 777, 779, 781, 783, 785, 787,789, 791, 793, 795, 797, 799, 801, 803, 805, 807, 809, 811, and 813,which has been subtracted using the polypeptides encoded by knownnucleotide sequences, including those of Genbank Accession numberCAA70664. Further details on immunological identification ofpolypeptides of the invention are found below. Additionally, fordistinguishing between duplexes with sequences of less than about 100nucleotides, a TMAC1 hybridization procedure known to those of ordinaryskill in the art can be used. See, e.g., Sorg, U. et al. Nucleic AcidsRes. (Sept. 11, 1991) 19(17), incorporated herein by reference in itsentirety for all purposes.

[0250] In one aspect, the invention provides a nucleic acid whichcomprises a unique subsequence in a nucleic acid selected from SEQ IDNO:1-5, 11-262,516-567, 620, 622, 624, 626, 628, 630, 632, 634, 636,638, 640, 642, 644, 646, 648, 650, 652, 654, 656, 658, 660, 662, 664,666, 668, 670, 672, 674, 676, 678, 680, 682, 684, 686, 688, 690, 692,694, 696, 698, 700, 702, 704, 706, 708, 710, 712, 714, 716, 718, 720,722, 724, 726, 728, 730, 732, 734, 736, 738, 740, 742, 744, 746, 748,750, 752, 754, 756, 758, 760, 762, 764, 766, 768, 770, 772, 774, 776,778, 780, 782, 784, 786, 788, 790, 792, 794, 796, 798, 800, 802, 804,806, 808, 810, and 812. The unique subsequence is unique as compared toa nucleic acid corresponding to any of Genbank Accession numbers Z99109and Y09476. Such unique subsequences can be determined by aligning anyof SEQ ID NO:1-5, 11-262, 516-567, 620, 622, 624, 626, 628, 630, 632,634, 636, 638, 640, 642, 644, 646, 648, 650, 652, 654, 656, 658, 660,662, 664, 666, 668, 670, 672, 674, 676, 678, 680, 682, 684, 686, 688,690, 692, 694, 696, 698, 700, 702, 704, 706, 708, 710, 712, 714, 716,718, 720, 722, 724, 726, 728, 730, 732, 734, 736, 738, 740, 742, 744,746, 748, 750, 752, 754, 756, 758, 760, 762, 764, 766, 768, 770, 772,774, 776, 778, 780, 782, 784, 786, 788, 790, 792, 794, 796, 798, 800,802, 804, 806, 808, 810, and 812 against the complete set of nucleicacids represented by GenBank accession numbers Z99109 and Y09476 orother related sequences available in public databases as of the filingdate of the subject application. Alignment can be performed using theBLAST algorithm set to default parameters. Any unique subsequence isuseful, e.g., as a probe to identify the nucleic acids of the invention.

[0251] Similarly, the invention includes a polypeptide which comprises aunique subsequence in a polypeptide selected from: SEQ ID NO:6-10,263-514, 568-619, 621, 623, 625, 627, 629, 631, 633, 635, 637, 639, 641,643, 645, 647, 649, 651, 653, 655, 657, 659, 661, 663, 665, 667, 669,671, 673, 675, 677, 679, 681, 683, 685, 687, 689, 691, 693,695,697,699,701,703, 705,707,709,711,713, 715,717,719,721,723,725,727,729, 731,733,735,737,739, 741,743,745,747,749,751,753,755,757,759, 761,763,765, 767, 769, 771, 773, 775, 777, 779,781, 783, 785, 787, 789, 791, 793, 795, 797, 799, 801, 803, 805, 807,809, 811, and 813. Here, the unique subsequence is unique as compared toa polypeptide corresponding to that of GenBank accession numberCAA70664. Here again, the polypeptide is aligned against the sequencesrepresented by accession number CAA70664. Note that if the sequencecorresponds to a non-translated sequence such as a pseudo gene, thecorresponding polypeptide is generated simply by in silico translationof the nucleic acid sequence into an amino acid sequence, where thereading frame is selected to correspond to the reading frame ofhomologous GAT polynucleotides.

[0252] The invention also provides for target nucleic acids whichhybridize under stringent conditions to a unique coding oligonucleotidewhich encodes a unique subsequence in a polypeptide selected from SEQ IDNO:6-10, 263-514, 568-619, 621, 623, 625, 627, 629, 631, 633, 635, 637,639, 641, 643, 645, 647, 649, 651, 653, 655, 657, 659, 661, 663, 665,667, 669, 671, 673, 675, 677, 679, 681, 683, 685, 687, 689, 691, 693,695,697,699,701,703, 705,707,709,711,713, 715,717,719,721,723,725,727,729, 731, 733, 735, 737, 739, 741, 743, 745, 747, 749, 751, 753,755, 757, 759, 761, 763, 765, 767, 769, 771, 773, 775, 777, 779, 781,783, 785, 787, 789, 791, 793, 795, 797, 799, 801, 803, 805, 807, 809,811, and 813, wherein the unique subsequence is unique as compared to apolypeptide corresponding to any of the control polypeptides. Uniquesequences are determined as noted above.

[0253] In one example, the stringent conditions are selected such that aperfectly complementary oligonucleotide to the coding oligonucleotidehybridizes to the coding oligonucleotide with at least about a 2.5×-10×higher, preferably at least about a 5-10× higher signal to noise ratiothan for hybridization of the perfectly complementary oligonucleotide toa control nucleic acid corresponding to any of the control polypeptides.Conditions can be selected such that higher ratios of signal to noiseare observed in the particular assay which is used, e.g., about 15×,20×, 30×, 50× or more. In this example, the target nucleic acidhybridizes to the unique coding oligonucleotide with at least a 2×higher signal to noise ratio as compared to hybridization of the controlnucleic acid to the coding oligonucleotide. Again, higher signal tonoise ratios can be selected, e.g., about 2.5×, 5×, 10×, 20×, 30×, 50×or more. The particular signal will depend on the label used in therelevant assay, e.g., a fluorescent label, a calorimetric label, aradioactive label, or the like.

[0254] Vectors, Promoters and Expression Systems,

[0255] The present invention also includes recombinant constructscomprising one or more of the nucleic acid sequences as broadlydescribed above. The constructs comprise a vector, such as, a plasmid, acosmid, a phage, a virus, a bacterial artificial chromosome (BAC), ayeast artificial chromosome (YAC), or the like, into which a nucleicacid sequence of the invention has been inserted, in a forward orreverse orientation. In a preferred aspect of this embodiment, theconstruct further comprises regulatory sequences, including, forexample, a promoter, operably linked to the sequence. Large numbers ofsuitable vectors and promoters are known to those of skill in the art,and are commercially available.

[0256] As previously discussed, general texts which describe molecularbiological techniques useful herein, including the use of vectors,promoters and many other relevant topics, include Berger and Kimmel,Guide to Molecular Cloning Techniques Methods in Enzymology Volume 152,Academic Press, Inc., San Diego, Calif. (“Berger”); Sambrook et al.,Molecular Cloning—A Laboratory Manual (2nd Ed.), Vol. 1-3, Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y., 1989 (“Sambrook”) andCurrent Protocols in Molecular Biology, F. M. Ausubel et al., eds.,Current Protocols, a joint venture between Greene Publishing Associates,Inc. and John Wiley & Sons, Inc., (supplemented through 1999)(“Ausubel”). Examples of protocols sufficient to direct persons of skillthrough in vitro amplification methods, including the polymerase chainreaction (PCR), the ligase chain reaction (LCR), Qβ-replicaseamplification and other RNA polymerase mediated techniques (e.g.,NASBA), e.g., for the production of the homologous nucleic acids of theinvention are found in Berger, Sambrook, and Ausubel, as well as inMullis et al., (1987) U.S. Pat. No. 4,683,202; PCR Protocols A Guide toMethods and Applications (Innis et al. eds.) Academic Press Inc. SanDiego, Calif. (1990) (“Innis”); Arnheim & Levinson (Oct. 1, 1990) C&EN36-47; The Journal Of NIH Research (1991) 3, 81-94; (Kwoh et al. (1989)Proc. Natl. Acad. Sci. USA 86, 1173; Guatelli et al. (1990) Proc. Natl.Acad. Sci. USA 87, 1874; Lomell et al. (1989) J. Clin. Chem 35, 1826;Landegren et al., (1988) Science 241, 1077-1080; Van Brunt (1990)Biotechnology 8, 291-294; Wu and Wallace, (1989) Gene 4:560; Barringeret al. (1990) Gene 89:117; and Sooknanan and Malek (1995) Biotechnology13: 563-564. Improved methods for cloning in vitro amplified nucleicacids are described in Wallace et al., U.S. Pat. No. 5,426,039. Improvedmethods for amplifying large nucleic acids by PCR are summarized inCheng et al. (1994) Nature 369: 684-685 and the references citedtherein, in which PCR amplicons of up to 40 kb are generated. One ofskill will appreciate that essentially any RNA can be converted into adouble stranded DNA suitable for restriction digestion, PCR expansionand sequencing using reverse transcriptase and a polymerase. See, e.g.,Ausubel, Sambrook and Berger, all supra.

[0257] The present invention also relates to engineered host cells thatare transduced (transformed or transfected) with a vector of theinvention (e.g., an invention cloning vector or an invention expressionvector), as well as the production of polypeptides of the invention byrecombinant techniques. The vector may be, for example, a plasmid, aviral particle, a phage, etc. The engineered host cells can be culturedin conventional nutrient media modified as appropriate for activatingpromoters, selecting transformants, or amplifying the GAT homologuegene. Culture conditions, such as temperature, pH and the like, arethose previously used with the host cell selected for expression, andwill be apparent to those skilled in the art and in the references citedherein, including, e.g., Sambrook, Ausubel and Berger, as well as e.g.,Freshney (1994) Culture of Animal Cells a Manual of Basic Technique,3^(rd) Ed., Wiley- Liss, New York and the references cited therein.

[0258] GAT polypeptides of the invention can be produced in non-animalcells such as plants, yeast, fungi, bacteria and the like. In additionto Sambrook, Berger and Ausubel, details regarding non-animal cellculture can be found in Payne et al. (1992) Plant Cell and TissueCulture in Liquid Systems, John Wiley & Sons, Inc. New York, N.Y.;Gamborg and Phillips (eds.) (1995) Plant Cell Tissue and Organ Culture;Fundamental Methods Springer Lab Manual, Springer-Verlag (Berlin,Heidelberg, N.Y.); and Atlas and Parks (eds.) The Handbook ofMicrobiological Media (1993) CRC Press, Boca Raton, Fla.

[0259] Polynucleotides of the present invention can be incorporated intoany one of a variety of expression vectors suitable for expressing apolypeptide. Suitable vectors include chromosomal, nonchromosomal andsynthetic DNA sequences, e.g., derivatives of SV40; bacterial plasmids;phage DNA; baculovirus; yeast plasmids; vectors derived fromcombinations of plasmids and phage DNA, viral DNA such as vaccinia,adenovirus, fowl pox virus, pseudorabies, adenovirus, adeno-associatedviruses, retroviruses and many others. Any vector that transducesgenetic material into a cell, and, if replication is desired, which isreplicable and viable in the relevant host can be used.

[0260] When incorporated into an expression vector, a polynucleotide ofthe invention is operatively linked to an appropriate transcriptioncontrol sequence (promoter) to direct mRNA synthesis. Examples of suchtranscription control sequences particularly suited for use intransgenic plants include the cauliflower mosaic virus (CaMV), figwortmosaic virus (FMV) and strawberry vein banding virus (SVBV) promoters,described in U.S. Provisional Application No. 60/245,354. Otherpromoters known to control expression of genes in prokaryotic oreukaryotic cells or their viruses and which can be used in someembodiments of the invention include SV40 promoter, E. coli lac or trppromoter, and the phage lambda P_(L) promoter. An expression vectoroptionally contains a ribosome binding site for translation initiation,and a transcription terminator, such as PinII. The vector alsooptionally includes appropriate sequences for amplifying expression,e.g., an enhancer.

[0261] In addition, the expression vectors of the present inventionoptionally contain one or more selectable marker genes to provide aphenotypic trait for selection of transformed host cells. Usually, theselectable marker gene will encode antibiotic or herbicide resistance.Suitable genes include those coding for resistance to the antibioticspectinomycin or streptomycin (e.g., the aada gene), the streptomycinphosphotransferase (SPT) gene coding for streptomycin resistance, theneomycin phosphotransferase (NPTII) gene encoding kanamycin or geneticinresistance, the hygromycin phosphotransferase (HPT) gene coding forhygromycin resistance. Additional selectable marker genes includedihydrofolate reductase or neomycin resistance for eukaryotic cellculture, and tetracycline or ampicillin resistance in E. coli.

[0262] Suitable genes coding for resistance to herbicides include thosewhich act to inhibit the action of acetolactate synthase (ALS), inparticular the sulfonylurea-type herbicides (e.g., the acetolactatesynthase (ALS) gene containing mutations leading to such resistance inparticular the S4 and/or Hra mutations), those which act to inhibit theaction of glutamine synthase, such as phosphinothricin or basta (e.g.,the bar gene), or other such genes known in the art. The bar geneencodes resistance to the herbicide basta and the ALS gene encodesresistance to the herbicide chlorsulfuron. In some instances, themodified GAT genes are used as selectable markers.

[0263] Vectors of the present invention can be employed to transform anappropriate host to permit the host to express an inventive protein orpolypeptide. Examples of appropriate expression hosts include: bacterialcells, such as E. coli, B. subtilis, Streptomyces, and Salmonellatyphimurium; fungal cells, such as Saccharomyces cerevisiae, Pichiapastoris, and Neurospora crassa; insect cells such as Drosophila andSpodoptera frugiperda; mammalian cells such as CHO, COS, BHK, HEK 293 orBowes melanoma; or plant cells or explants, etc. It is understood thatnot all cells or cell lines need to be capable of producing fullyfunctional GAT polypeptides; for example, antigenic fragments of a GATpolypeptide may be produced. The present invention is not limited by thehost cells employed.

[0264] In bacterial systems, a number of expression vectors may beselected depending upon the use intended for the GAT polypeptide. Forexample, when large quantities of GAT polypeptide or fragments thereofare needed for commercial production or for induction of antibodies,vectors which direct high level expression of fusion proteins that arereadily purified can be desirable. Such vectors include, but are notlimited to, multifunctional E. coli cloning and expression vectors suchas BLUESCRIPT (Stratagene), in which the GAT polypeptide coding sequencemay be ligated into the vector in-frame with sequences for theamino-terminal Met and the subsequent 7 residues of beta-galactosidaseso that a hybrid protein is produced; pIN vectors (Van Heeke & Schuster(1989) J Biol Chem 264:5503-5509); pET vectors (Novagen, Madison Wis.);and the like.

[0265] Similarly, in the yeast Saccharomyces cerevisiae a number ofvectors containing constitutive or inducible promoters such as alphafactor, alcohol oxidase and PGH may be used for production of the GATpolypeptides of the invention. For reviews, see Ausubel (supra) andGrant et al. (1987) Methods in Enzymology 153:516-544.

[0266] In mammalian host cells, a variety of expression systems,including viral-based systems, may be utilized. In cases where anadenovirus is used as an expression vector, a coding sequence, e.g., ofa GAT polypeptide, is optionally ligated into an adenovirustranscription/translation complex consisting of the late promoter andtripartite leader sequence. Insertion of a GAT polypeptide coding regioninto a nonessential E1 or E3 region of the viral genome will result in aviable virus capable of expressing a GAT in infected host cells (Loganand Shenk (1984) Proc Natl Acad Sci USA 81:3655-3659). In addition,transcription enhancers, such as the rous sarcoma virus (RSV) enhancer,may be used to increase expression in mammalian host cells.

[0267] Similarly, in plant cells, expression can be driven from atransgene integrated into a plant chromosome, or cytoplasmically from anepisomal or viral nucleic acid. In the case of stably integratedtransgenes, it is often desirable to provide sequences capable ofdriving constitutive or inducible expression of the GAT polynucleotidesof the invention, for example, using viral, e.g., CaMV, or plant derivedregulatory sequences. Numerous plant derived regulatory sequences havebeen described, including sequences which direct expression in a tissuespecific manner, e.g., TobRB7, patatin B33, GRP gene promoters, therbcS-3A promoter, and the like. Alternatively, high level expression canbe achieved by transiently expressing exogenous sequences of a plantviral vector, e.g., TMV, BMV, etc. Typically, transgenic plantsconstitutively expressing a GAT polynucleotide of the invention will bepreferred, and the regulatory sequences are selected to insureconstitutive stable expression of the GAT polypeptide.

[0268] Typical vectors useful for expression of nucleic acids in higherplants are well known in the art and include vectors derived from thetumor-inducing (Ti) plasmid of Agrobacterium tumefaciens described byRogers et al., Meth. In Enzymol., 153:253-277 (1987). Exemplary A.tumefaciens vectors useful herein are plasmids pKYLX6 and pKYLX7 ofSchardl et al., Gene, 61:1-11 (1987) and Berger et al., Proc. Natl.Acad. Sci. U.S.A., 86:8402-8406 (1989). Another useful vector herein isplasmid pBI101.2 that is available from Clontech Laboratories, Inc.(Palo Alto, Calif.). A variety of plant viruses that can be employed asvectors are known in the art and include cauliflower mosaic virus(CaMV), geminivirus, brome mosaic virus, and tobacco mosaic virus.

[0269] In some embodiments of the present invention, a GATpolynucleotide construct suitable for transformation of plant cells isprepared. For example, a desired GAT polynucleotide can be incorporatedinto a recombinant expression cassette to facilitate introduction of thegene into a plant and subsequent expression of the encoded polypeptide.An expression cassette will typically comprise a GAT polynucleotide, orfunctional fragment thereof, operably linked to a promoter sequence andother transcriptional and translational initiation regulatory sequenceswhich will direct expression of the sequence in the intended tissues(e.g., entire plant, leaves, seeds) of the transformed plant.

[0270] For example, a strongly or weakly constitutive plant promoter canbe employed which will direct expression of the GAT polypeptide in alltissues of a plant. Such promoters are active under most environmentalconditions and states of development or cell differentiation. Examplesof constitutive promoters include the cauliflower mosaic virus (CaMV)35S transcription initiation region, the 1′- or 2′- promoter derivedfrom T-DNA of Agrobacterium tumefaciens, the ubiquitin 1 promoter, theSmas promoter, the cinnamyl alcohol dehydrogenase promoter (U.S. Pat.No. 5,683,439), the Nos promoter, the pEmu promoter, the rubiscopromoter, the GRP1-8 promoter and other transcription initiation regionsfrom various plant genes known to those of skill. In situations in whichover expression of a GAT polynucleotide is detrimental to the plant orotherwise undesirable, one of skill, upon review of this disclosure,will recognize that weak constitutive promoters can be used forlow-levels of expression. In those cases where high levels of expressionis not harmful to the plant, a strong promoter, e.g., a t-RNA or otherpol III promoter, or a strong pol II promoter, such as the cauliflowermosaic virus promoter, can be used.

[0271] Alternatively, a plant promoter may be under environmentalcontrol. Such promoters are referred to here as “inducible” promoters.Examples of environmental conditions that may effect transcription byinducible promoters include pathogen attack, anaerobic conditions, orthe presence of light. In particular, examples of inducible promotersare the Adh1 promoter which is inducible by hypoxia or cold stress, theHsp70 promoter which is inducible by heat stress, and the PPDK promoterwhich is inducible by light. Also useful are promoters which arechemically inducible.

[0272] The promoters used in the present invention can be“tissue-specific” and, as such, under developmental control in that thepolynucleotide is expressed only in certain tissues, such as leaves,roots, fruit, flowers and/or seeds. An exemplary promoter is the antherspecific promoter 5126 (U.S. Pat. Nos. 5,689,049 and 5,689,051).Examples of seed-preferred promoters include, but are not limited to, 27kD gamma zein promoter and waxy promoter, Boronat et al. Plant Sci. 47,95-102 (1986); Reina et al. Nucleic Acids Res. 18 (21), 6426 (1990); andKloesgen et al., Mol. Gen. Genet. 203, 237-244 (1986). Promoters thatexpress in the embryo, pericarp, and endosperm are disclosed in U.S.patent application Ser. Nos. 60/097,233 filed Aug. 20, 1998 and60/098,230 filed Aug. 28, 1998. The disclosures each of these areincorporated herein by reference in their entirety. In embodiments inwhich one or more nucleic acid sequences endogenous to the plant systemare incorporated into the construct, the endogenous promoters (orvariants thereof) from these genes can be employed for directingexpression of the genes in the transfected plant. Tissue-specificpromoters can also be used to direct expression of heterologouspolynucleotides.

[0273] In general, the particular promoter used in the expressioncassette in plants depends on the intended application. Eitherheterologous or non-heterologous (i.e., endogenous) promoters can beemployed to direct expression of the nucleic acids of the presentinvention. These promoters can also be used, for example, in expressioncassettes to drive expression of antisense nucleic acids to reduce,increase, or alter the concentration and/or composition of the proteinsof the present invention in a desired tissue. Any of a number ofpromoters which direct transcription in plant cells are suitable. Thepromoter can be either constitutive or inducible. In addition to thepromoters noted above, promoters of bacterial origin which operate inplants include the octopine synthase promoter, the nopaline synthasepromoter and other promoters derived from native Ti plasmids (see,Herrara-Estrella et al. (1983) Nature 303:209-213). Viral promotersinclude the 35S and 19S RNA promoters of cauliflower mosaic virus (Odellet al. (1985) Nature 313:810-812). Other plant promoters include theribulose-1,3-bisphosphate carboxylase small subunit promoter and thephaseolin promoter. The promoter sequence from the E8 gene and othergenes may also be used. The isolation and sequence of the E8 promoter isdescribed in detail in Deikman and Fischer (1988) EMBO J. 7:3315-3327.

[0274] To identify candidate promoters, the 5′ portions of a genomicclone is analyzed for sequences characteristic of promoter sequences.For instance, promoter sequence elements include the TATA box consensussequence (TATAAT), which is usually 20 to 30 base pairs upstream of thetranscription start site. In plants, further upstream from the TATA box,at positions −80 to −100, there is typically a promoter element with aseries of adenines surrounding the trinucleotide G (or T) as describedby Messing et al. (1983) Genetic Engineering in Plants, Kosage, et al.(eds.), pp. 221-227.

[0275] In preparing polynucleotide constructs, e.g., vectors, of theinvention, sequences other than the promoter and the cojoinedpolynucleotide can also be employed. If normal polypeptide expression isdesired, a polyadenylation region at the 3′-end of a GAT-encoding regioncan be included. The polyadenylation region can be derived, for example,from a variety of plant genes, or from T-DNA. The 3′ end sequence to beadded can be derived from, for example, the nopaline synthase oroctopine synthase genes, or alternatively from another plant gene, orless preferably from any other eukaryotic gene.

[0276] An intron sequence can be added to the 5′ untranslated region ofthe coding sequence or the partial coding sequence to increase theamount of the mature message that accumulates. See for example Buchmanand Berg, Mol. Cell Biol. 8:4395-4405 (1988) and Callis et al., GenesDev. 1:1183-1200 (1987). Use of maize introns Adh1, intron 1, 2, and 6,and the Bronze-1 intron are known in the art. See generally, The MaizeHandbook, Chapter 116, Freeling and Walbot, eds., Springer, N.Y. (1994).

[0277] The construct can also include a marker gene which confers aselectable phenotype on plant cells. For example, the marker may encodebiocide tolerance, particularly antibiotic tolerance, such as toleranceto kanamycin, G418, bleomycin, hygromycin, or herbicide tolerance, suchas tolerance to chlorosulfuron, or phosphinothricin (the activeingredient in the herbicides bialaphos and Basta).

[0278] Specific initiation signals can aid in efficient translation of aGAT polynucleotide-encoding sequence of the present invention. Thesesignals can include, e.g., the ATG initiation codon and adjacentsequences. In cases where a GAT polypeptide-encoding sequence, itsinitiation codon and upstream sequences are inserted into an appropriateexpression vector, no additional translational control signals may beneeded. However, in cases where only the coding sequence (e.g., a matureprotein coding sequence), or a portion thereof, is inserted, exogenoustranscriptional control signals including the initiation codon must beprovided. Furthermore, the initiation codon must be in the correctreading frame to ensure transcription of the entire insert. Exogenoustranscriptional elements and initiation codons can be of variousorigins, both natural and synthetic. The efficiency of expression may beenhanced by the inclusion of enhancers appropriate to the cell system inuse (Scharf et al. (1994) Results Probl Cell Differ 20:125-62 andBittner et al. (1987) Methods in Enzymol 153:516-544).

[0279] Secretion/Localization Sequences

[0280] Polynucleotides of the invention can also be fused, for example,in-frame to nucleic acids encoding a secretion/localization sequence, totarget polypeptide expression to a desired cellular compartment,membrane, or organelle of a host cell, or to direct polypeptidesecretion to the periplasmic space or into the cell culture media. Suchsequences are known to those of skill, and include secretion leaderpeptides, organelle targeting sequences (e.g., nuclear localizationsequences, ER retention signals, mitochondrial transit sequences, andchloroplast transit sequences), membrane localization/anchor sequences(e.g., stop transfer sequences, GPI anchor sequences), and the like.

[0281] In a preferred embodiment, a polynucleotide of the invention isfused in frame with an N-terminal chloroplast transit sequence (orchloroplast transit peptide sequence) derived from a gene encoding apolypeptide that is normally targeted to the chloroplast. Such sequencesare typically rich in serine and threonine; are deficient in aspartate,glutamate, and tyrosine; and generally have a central domain rich inpositively charged amino acids.

[0282] Expression Hosts

[0283] In a further embodiment, the present invention relates to hostcells containing the above-described constructs. The host cell can be aeukaryotic cell, such as a mammalian cell, a yeast cell, or a plantcell, or the host cell can be a prokaryotic cell, such as a bacterialcell. Introduction of the construct into the host cell can be effectedby calcium phosphate transfection, DEAE-Dextran mediated transfection,electroporation, or other common techniques (Davis et al., Basic Methodsin Molecular Biology).

[0284] A host cell is optionally chosen for its ability to modulate theexpression of the inserted sequences or to process the expressed proteinin the desired fashion. Such modifications of the protein include, butare not limited to, acetylation, carboxylation, glycosylation,phosphorylation, lipidation and acylation. Post-translational processingthat cleaves a “pre” or a “prepro” form of the protein may also beimportant for correct insertion, folding and/or function. Different hostcells such as E. coli, Bacillus sp., yeast or mammalian cells such asCHO, HeLa, BHK, MDCK, 293, WI38, etc. have specific cellular machineryand characteristic mechanisms, e.g., for post-translational activitiesand may be chosen to ensure the desired modification and processing ofthe introduced, foreign protein.

[0285] For long-term, high-yield production of recombinant proteins,stable expression systems can be used. For example, plant cells,explants or tissues, e.g. shoots, or leaf discs, which stably express apolypeptide of the invention are transduced using expression vectorswhich contain viral origins of replication or endogenous expressionelements and a selectable marker gene. Following the introduction of thevector, cells may be allowed to grow for a period determined to beappropriate for the cell type, e.g., 1 or more hours for bacterialcells, 1-4 days for plant cells, 2-4 weeks for some plant explants, inan enriched media before they are switched to selective media. Thepurpose of the selectable marker is to confer resistance to selection,and its presence allows growth and recovery of cells which successfullyexpress the introduced sequences. For example, transgenic plantsexpressing the polypeptides of the invention can be selected directlyfor resistance to the herbicide, glyphosate. Resistant embryos derivedfrom stably transformed explants can be proliferated, e.g., using tissueculture techniques appropriate to the cell type.

[0286] Host cells transformed with a nucleotide sequence encoding apolypeptide of the invention are optionally cultured under conditionssuitable for the expression and recovery of the encoded protein fromcell culture. The protein or fragment thereof produced by a recombinantcell may be secreted, membrane-bound, or contained intracellularly,depending on the sequence and/or the vector used. As will be understoodby those of skill in the art, expression vectors containing GATpolynucleotides of the invention can be designed with signal sequenceswhich direct secretion of the mature polypeptides through a prokaryoticor eukaryotic cell membrane.

[0287] Additional Polypeptide Sequences

[0288] Polynucleotides of the present invention may also comprise acoding sequence fused in-frame to a marker sequence that, e.g.,facilitates purification of the encoded polypeptide. Such purificationfacilitating domains include, but are not limited to, metal chelatingpeptides such as histidine-tryptophan modules that allow purification onimmobilized metals, a sequence which binds glutathione (e.g., GST), ahemagglutinin (HA) tag (corresponding to an epitope derived from theinfluenza hemagglutinin protein; Wilson et al. (1984) Cell 37:767),maltose binding protein sequences, the FLAG epitope utilized in theFLAGS extension/affinity purification system (Immunex Corp, Seattle,Wash.), and the like. The inclusion of a protease-cleavable polypeptidelinker sequence between the purification domain and the GAT homologuesequence is useful to facilitate purification. One expression vectorcontemplated for use in the compositions and methods described hereinprovides for expression of a fusion protein comprising a polypeptide ofthe invention fused to a polyhistidine region separated by anenterokinase cleavage site. The histidine residues facilitatepurification on IMIAC (immobilized metal ion affinity chromatography, asdescribed in Porath et al. (1992) Protein Expression and Purification3:263-281) while the enterokinase cleavage site provides a means forseparating the GAT homologue polypeptide from the fusion protein. pGEXvectors (Promega; Madison, Wis.) may also be used to express foreignpolypeptides as fusion proteins with glutathione S-transferase (GST). Ingeneral, such fusion proteins are soluble and can easily be purifiedfrom lysed cells by adsorption to ligand-agarose beads (e.g.,glutathione-agarose in the case of GST-fusions) followed by elution inthe presence of free ligand.

[0289] Polypeptide Production and Recovery

[0290] Following transduction of a suitable host and growth of the hostcells to an appropriate cell density, the selected promoter is inducedby appropriate means (e.g., temperature shift or chemical induction) andcells are cultured for an additional period. Cells are typicallyharvested by centrifugation, disrupted by physical or chemical means,and the resulting crude extract retained for further purification.Microbial cells employed in the expression of proteins can be disruptedby any convenient method, including freeze-thaw cycling, sonication,mechanical disruption, or use of cell lysing agents, or other methods,which are well known to those skilled in the art.

[0291] As noted, many references are available for the culture andproduction of many cells, including cells of bacterial, plant, animal(especially mammalian) and archebacterial origin. See e.g., Sambrook,Ausubel, and Berger (all supra), as well as Freshney (1994) Culture ofAnimal Cells, a Manual of Basic Technique, 3^(rd) Ed., Wiley-Liss, NewYork and the references cited therein; Doyle and Griffiths (1997)Mammalian Cell Culture: Essential Techniques John Wiley and Sons, NY;Humason (1979) Animal Tissue Techniques, 4^(th) Ed. W. H. Freeman andCompany; and Ricciardelli, et al., (1989) In vitro Cell Dev. Biol.25:1016-1024. For plant cell culture and regeneration see, Payne et al.(1992) Plant Cell and Tissue Culture in Liquid Systems John Wiley &Sons, Inc. New York, N.Y.; Gamborg and Phillips (eds.) (1995) PlantCell, Tissue and Organ Culture; Fundamental Methods Springer Lab Manual,Springer-Verlag (Berlin, Heidelberg, N.Y.); Jones, ed. (1984) Plant GeneTransfer and Expression Protocols, Humana Press, Totowa, N.J.; and PlantMolecular Biology (1993) R. R. D. Croy, ed. Bios Scientific Publishers,Oxford, U.K. ISBN 0 12 198370 6. Cell culture media in general are setforth in Atlas and Parks (eds.) The Handbook of Microbiological Media(1993) CRC Press, Boca Raton, Fla. Additional information for cellculture is found in available commercial literature such as the LifeScience Research Cell Culture Catalogue (1998) from Sigma-Aldrich, Inc.(St Louis, Mo.) (“Sigma-LSRCCC”) and, e.g., The Plant Culture Catalogueand supplement (1997) also from Sigma-Aldrich, Inc. (St Louis, Mo.)(“Sigma-PCCS”). Further details regarding plant cell transformation andtransgenic plant production are found below.

[0292] Polypeptides of the invention can be recovered and purified fromrecombinant cell cultures by any of a number of methods well known inthe art, including ammonium sulfate or ethanol precipitation, acidextraction, anion or cation exchange chromatography, phosphocellulosechromatography, hydrophobic interaction chromatography, affinitychromatography (e.g., using any of the tagging systems noted herein),hydroxylapatite chromatography, and lectin chromatography. Proteinrefolding steps can be used, as desired, in completing the configurationof the mature protein. Finally, high performance liquid chromatography(HPLC) can be employed in the final purification steps. In addition tothe references noted supra, a variety of purification methods are wellknown in the art, including, e.g., those set forth in Sandana (1997)Bioseparation of Proteins, Academic Press, Inc.; Bollag et al. (1996)Protein Methods 2^(nd) Ed. Wiley-Liss, NY; Walker (1996) The ProteinProtocols Handbook Humana Press, NJ, Harris and Angal (1990) ProteinPurification Applications: A Practical Approach IRL Press at Oxford,Oxford, England; Harris and Angal Protein Purification Methods: APractical Approach IRL Press at Oxford, Oxford, England; Scopes (1993)Protein Purification: Principles and Practice 3^(rd) Ed. SpringerVerlag, NY; Janson and Ryden (1998) Protein Purification: PrinciplesHigh Resolution Methods and Applications 2^(nd) Ed. Wiley-VCH, NY; andWalker (1998) Protein Protocols on CD-ROM Humana Press, NJ.

[0293] In some cases, it is desirable to produce the GAT polypeptide ofthe invention in a large scale suitable for industrial and/or commercialapplications. In such cases bulk fermentation procedures are employed.Briefly, a GAT polynucleotide, e.g., a polynucleotide comprising any oneof SEQ ID NO: 1-5, 11-262, 516-567, 620, 622, 624, 626, 628, 630, 632,634, 636, 638, 640, 642, 644, 646, 648, 650, 652, 654, 656, 658, 660,662, 664, 666, 668, 670, 672, 674, 676, 678, 680, 682, 684, 686, 688,690, 692, 694, 696, 698, 700, 702, 704, 706, 708, 710, 712, 714, 716,718, 720, 722, 724, 726, 728, 730, 732, 734, 736, 738, 740, 742, 744,746, 748, 750, 752, 754, 756, 758, 760, 762, 764, 766, 768, 770, 772,774, 776, 778, 780, 782, 784, 786, 788, 790, 792, 794, 796, 798, 800,802, 804, 806, 808, 810, and 812, or other nucleic acids encoding GATpolypeptides of the invention can be cloned into an expression vector.For example, U.S. Pat. No. 5,955,310 to Widner et al. “METHODS FORPRODUCING A POLYPEPTIDE IN A BACILLUS CELL,” describes a vector withtandem promoters, and stabilizing sequences operably linked to apolypeptide encoding sequence. After inserting the polynucleotide ofinterest into a vector, the vector is transformed into a bacterial,e.g., a Bacillus subtilis strain PL1801IIE (amyE, apr, npr,spoIIE::Tn917) host. The introduction of an expression vector into aBacillus cell may, for instance, be effected by protoplasttransformation (see, e.g., Chang and Cohen (1979) Molecular GeneralGenetics 168:111), by using competent cells (see, e.g., Young andSpizizin (1961) Journal of Bacteriology 81:823, or Dubnau andDavidoff-Abelson (1971) Journal of Molecular Biology 56:209), byelectroporation (see, e.g., Shigekawa and Dower (1988) Biotechniques6:742), or by conjugation (see, e.g., Koehler and Thorne (1987) Journalof Bacteriology 169:5271), see also, Ausubel, Sambrook and Berger, allsupra.

[0294] The transformed cells are cultivated in a nutrient mediumsuitable for production of the polypeptide using methods that are knownin the art. For example, the cell may be cultivated by shake flaskcultivation, small-scale or large-scale fermentation (includingcontinuous, batch, fed-batch, or solid state fermentations) inlaboratory or industrial fermentors performed in a suitable medium andunder conditions allowing the polypeptide to be expressed and/orisolated. The cultivation takes place in a suitable nutrient mediumcomprising carbon and nitrogen sources and inorganic salts, usingprocedures known in the art. Suitable media are available fromcommercial suppliers or may be prepared according to publishedcompositions (e.g., in catalogues of the American Type CultureCollection). The secreted polypeptide can be recovered directly from themedium.

[0295] The resulting polypeptide may be isolated by methods known in theart. For example, the polypeptide may be isolated from the nutrientmedium by conventional procedures including, but not limited to,centrifugation, filtration, extraction, spray-drying, evaporation, orprecipitation. The isolated polypeptide may then be further purified bya variety of procedures known in the art including, but not limited to,chromatography (e.g., ion exchange, affinity, hydrophobic,chromatofocusing, and size exclusion), electrophoretic procedures (e.g.,preparative isoelectric focusing), differential solubility (e.g.,ammonium sulfate precipitation), or extraction (see, e.g., Bollag et al.(1996) Protein Methods, 2^(nd) Ed. Wiley-Liss, NY and Walker (1996) TheProtein Protocols Handbook Humana Press, NJ).

[0296] Cell-free transcription/translation systems can also be employedto produce polypeptides using DNAs or RNAs of the present invention.Several such systems are commercially available. A general guide to invitro transcription and translation protocols is found in Tymms (1995)In vitro Transcription and Translation Protocols: Methods in MolecularBiology Volume 37, Garland Publishing, NY.

[0297] Substrates and Formats for Sequence Recombination

[0298] The polynucleotides of the invention are optionally used assubstrates for a variety of diversity generating procedures, e.g.,mutation, recombination and recursive recombination reactions, inaddition to their use in standard cloning methods as set forth in, e.g.,Ausubel, Berger and Sambrook, to produce additional GAT polynucleotidesand polypeptides with desired properties. A variety of diversitygenerating protocols are available and described in the art. Theprocedures can be used separately, and/or in combination to produce oneor more variants of a polynucleotide or set of polynucleotides, as wellvariants of encoded proteins. Individually and collectively, theseprocedures provide robust, widely applicable ways of generatingdiversified polynucleotides and sets of polynucleotides (including,e.g., polynucleotide libraries) useful, e.g., for the engineering orrapid evolution of polynucleotides, proteins, pathways, cells and/ororganisms with new and/or improved characteristics. The process ofaltering the sequence can result in, for example, single nucleotidesubstitutions, multiple nucleotide substitutions, and insertion ordeletion of regions of the nucleic acid sequence.

[0299] While distinctions and classifications are made in the course ofthe ensuing discussion for clarity, it will be appreciated that thetechniques are often not mutually exclusive. Indeed, the various methodscan be used singly or in combination, in parallel or in series, toaccess diverse sequence variants.

[0300] The result of any of the diversity generating proceduresdescribed herein can be the generation of one or more polynucleotides,which can be selected or screened for polynucleotides that encodeproteins with or which confer desirable properties. Followingdiversification by one or more of the methods described herein, orotherwise available to one of skill, any polynucleotides that areproduced can be selected for a desired activity or property, e.g.altered K_(m) for glyphosate, altered K_(m) for acetyl CoA, use ofalternative cofactors (e.g., propionyl CoA) increased k_(cat), etc. Thiscan include identifying any activity that can be detected, for example,in an automated or automatable format, by any of the assays in the art.For example, GAT homologs with increased specific activity can bedetected by assaying the conversion of glyphosate to N-acetylglyphosate,e.g., by mass spectrometry. Alternatively, improved ability to conferresistance to glyphosate can be assayed by growing bacteria transformedwith a nucleic acid of the invention on agar containing increasingconcentrations of glyphosate or by spraying transgenic plantsincorporating a nucleic acid of the invention with glyphosate. A varietyof related (or even unrelated) properties can be evaluated, in serial orin parallel, at the discretion of the practitioner. Additional detailsregarding recombination and selection for herbicide tolerance can befound, e.g., in “DNA SHUFFLING TO PRODUCE HERBICIDE RESISTANT CROPS”(U.S. Pub. No. 2002/0058249) filed Aug. 12,1999.

[0301] Descriptions of a variety of diversity generating procedures,including multigene shuffling and methods for generating modifiednucleic acid sequences encoding multiple enzymatic domains, are foundthe following publications and the references cited therein: Soong, N.et al. (2000) “Molecular breeding of viruses” Nat Genet 25(4):436-39;Stemmer, et al. (1999) “Molecular breeding of viruses for targeting andother clinical properties” Tumor Targeting 4:1-4; Ness et al. (1999)“DNA Shuffling of subgenomic sequences of subtilisin” NatureBiotechnology 17:893-896; Chang et al. (1999) “Evolution of a cytokineusing DNA family shuffling” Nature Biotechnology 17:793-797; Minshulland Stemmer (1999) “Protein evolution by molecular breeding” CurrentOpinion in Chemical Biology 3:284-290; Christians et al. (1999)“Directed evolution of thymidine kinase for AZT phosphorylation usingDNA family shuffling” Nature Biotechnology 17:259-264; Crameri et al.(1998) “DNA shuffling of a family of genes from diverse speciesaccelerates directed evolution” Nature 391:288-291; Crameri et al.(1997) “Molecular evolution of an arsenate detoxification pathway by DNAshuffling,” Nature Biotechnology 15:436-438; Zhang et al. (1997)“Directed evolution of an effective fucosidase from a galactosidase byDNA shuffling and screening” Proc. Natl. Acad. Sci. USA 94:4504-4509;Patten et al. (1997) “Applications of DNA Shuffling to Pharmaceuticalsand Vaccines” Current Opinion in Biotechnology 8:724-733; Crameri et al.(1996) “Construction and evolution of antibody-phage libraries by DNAshuffling” Nature Medicine 2:100-103; Crameri et al. (1996) “Improvedgreen fluorescent protein by molecular evolution using DNA shuffling”Nature Biotechnology 14:315-319; Gates et al. (1996) “Affinity selectiveisolation of ligands from peptide libraries through display on a lacrepressor ‘headpiece dimer’” Journal of Molecular Biology 255:373-386;Stemmer (1996) “Sexual PCR and Assembly PCR” In: The Encyclopedia ofMolecular Biology. VCH Publishers, New York. pp.447-457; Crameri andStemmer (1995) “Combinatorial multiple cassette mutagenesis creates allthe permutations of mutant and wildtype cassettes” BioTechniques18:194-195; Stemmer et al., (1995) “Single-step assembly of a gene andentire plasmid from large numbers of oligodeoxy-ribonucleotides” Gene,164:49-53; Stemmer (1995) “The Evolution of Molecular Computation”Science 270:1510; Stemmer (1995) “Searching Sequence Space”Bio/Technology 13:549-553; Stemmer (1994) “Rapid evolution of a proteinin vitro by DNA shuffling” Nature 370:389-391; and Stemmer (1994) “DNAshuffling by random fragmentation and reassembly: In vitro recombinationfor molecular evolution.” Proc. Natl. Acad. Sci. USA 91:10747-10751.

[0302] Mutational methods of generating diversity include, for example,site-directed mutagenesis (Ling et al. (1997) “Approaches to DNAmutagenesis: an overview” Anal Biochem. 254(2): 157-178; Dale et al.(1996) “Oligonucleotide-directed random mutagenesis using thephosphorothioate method” Methods Mol. Biol. 57:369-374; Smith (1985) “Invitro mutagenesis” Ann. Rev. Genet. 19:423-462; Botstein & Shortle(1985) “Strategies and applications of in vitro mutagenesis” Science229:1193-1201; Carter (1986) “Site-directed mutagenesis” Biochem. J.237:1-7; and Kunkel (1987) “The efficiency of oligonucleotide directedmutagenesis” in Nucleic Acids & Molecular Biology (Eckstein, F. andLilley, D. M. J. eds., Springer Verlag, Berlin)); mutagenesis usinguracil containing templates (Kunkel (1985) “Rapid and efficientsite-specific mutagenesis without phenotypic selection” Proc. Natl.Acad. Sci. USA 82:488-492; Kunkel et al. (1987) “Rapid and efficientsite-specific mutagenesis without phenotypic selection” Methods inEnzymol. 154, 367-382; and Bass et al. (1988) “Mutant Trp repressorswith new DNA-binding specificities” Science 242:240-245);oligonucleotide-directed mutagenesis (Methods in Enzymol. 100: 468-500(1983); Methods in Enzymol. 154: 329-350 (1987); Zoller & Smith (1982)“Oligonucleotide-directed mutagenesis using M13-derived vectors: anefficient and general procedure for the production of point mutations inany DNA fragment” Nucleic Acids Res. 10:6487-6500; Zoller & Smith (1983)“Oligonucleotide-directed mutagenesis of DNA fragments cloned into M13vectors” Methods in Enzymol. 100:468-500; and Zoller & Smith (1987)“Oligonucleotide-directed mutagenesis: a simple method using twooligonucleotide primers and a single-stranded DNA template” Methods inEnzymol. 154:329-350); phosphorothioate-modified DNA mutagenesis (Tayloret al. (1985) “The use of phosphorothioate-modified DNA in restrictionenzyme reactions to prepare nicked DNA” Nucl. Acids Res. 13: 8749-8764;Taylor et al. (1985) “The rapid generation of oligonucleotide-directedmutations at high frequency using phosphorothioate-modified DNA” Nucl.Acids Res. 13: 8765-8787; Nakamaye & Eckstein (1986) “Inhibition ofrestriction endonuclease Nci I cleavage by phosphorothioate groups andits application to oligonucleotide-directed mutagenesis” Nucl. AcidsRes. 14: 9679-9698; Sayers et al. (1988) “Y-T Exonucleases inphosphorothioate-based oligonucleotide-directed mutagenesis” Nucl. AcidsRes. 16:791-802; and Sayers et al. (1988) “Strand specific cleavage ofphosphorothioate-containing DNA by reaction with restrictionendonucleases in the presence of ethidium bromide” Nucl. Acids Res. 16:803-814); mutagenesis using gapped duplex DNA (Kramer et al. (1984) “Thegapped duplex DNA approach to oligonucleotide-directed mutationconstruction” Nucl. Acids Res. 12: 9441-9456; Kramer & Fritz (1987)Methods in Enzymol. “Oligonucleotide-directed construction of mutationsvia gapped duplex DNA” 154:350-367; Kramer et al. (1988) “Improvedenzymatic in vitro reactions in the gapped duplex DNA approach tooligonucleotide-directed construction of mutations” Nucl. Acids Res. 16:7207; and Fritz et al. (1988) “Oligonucleotide-directed construction ofmutations: a gapped duplex DNA procedure without enzymatic reactions invitro” Nucl. Acids Res. 16: 6987-6999).

[0303] Additional suitable methods include point mismatch repair (Krameret al. (1984) “Point Mismatch Repair” Cell 38:879-887), mutagenesisusing repair-deficient host strains (Carter et al. (1985) “Improvedoligonucleotide site-directed mutagenesis using M13 vectors” Nucl. AcidsRes. 13: 4431-4443; and Carter (1987) “Improved oligonucleotide-directedmutagenesis using M13 vectors” Methods in Enzymol. 154: 382-403),deletion mutagenesis (Eghtedarzadeh & Henikoff (1986) “Use ofoligonucleotides to generate large deletions” Nucl. Acids Res. 14:5115), restriction-selection and restriction-purification (Wells et al.(1986) “Importance of hydrogen-bond formation in stabilizing thetransition state of subtilisin” Phil. Trans. R. Soc. Lond. A 317:415-423), mutagenesis by total gene synthesis (Nambiar et al. (1984)“Total synthesis and cloning of a gene coding for the ribonuclease Sprotein” Science 223: 1299-1301; Sakamar and Khorana (1988) “Totalsynthesis and expression of a gene for the a-subunit of bovine rod outersegment guanine nucleotide-binding protein (transducin)” Nucl. AcidsRes. 14: 6361-6372; Wells et al. (1985) “Cassette mutagenesis: anefficient method for generation of multiple mutations at defined sites”Gene 34:315-323; and Grundström et al. (1985) “Oligonucleotide-directedmutagenesis by microscale ‘shot-gun’ gene synthesis” Nucl. Acids Res.13: 3305-3316); double-strand break repair (Mandecki (1986); Arnold(1993) “Protein engineering for unusual environments” Current Opinion inBiotechnology 4:450-455; and “Oligonucleotide-directed double-strandbreak repair in plasmids of Escherichia coli: a method for site-specificmutagenesis” Proc. Natl. Acad. Sci. USA, 83:7177-7181). Additionaldetails on many of the above methods can be found in Methods inEnzymology Volume 154, which also describes useful controls fortrouble-shooting problems with various mutagenesis methods.

[0304] Additional details regarding various diversity generating methodscan be found in the following U.S. patents, PCT publications, and EPOpublications: U.S. Pat. No. 5,605,793 to Stemmer (Feb. 25, 1997),“Methods for In Vitro Recombination;” U.S. Pat. No. 5,811,238 to Stemmeret al. (Sep. 22, 1998) “Methods for Generating Polynucleotides havingDesired Characteristics by Iterative Selection and Recombination;” U.S.Pat. No. 5,830,721 to Stemmer et al. (Nov. 3, 1998), “DNA Mutagenesis byRandom Fragmentation and Reassembly;” U.S. Pat. No. 5,834,252 toStemmer, et al. (Nov. 10, 1998) “End-Complementary Polymerase Reaction;”U.S. Pat. No. 5,837,458 to Minshull, et al. (Nov. 17, 1998), “Methodsand Compositions for Cellular and Metabolic Engineering;” WO 95/22625,Stemmer and Crameri, “Mutagenesis by Random Fragmentation andReassembly;” WO 96/33207 by Stemmer and Lipschutz “End ComplementaryPolymerase Chain Reaction;” WO 97/20078 by Stemmer and Crameri “Methodsfor Generating Polynucleotides having Desired Characteristics byIterative Selection and Recombination;” WO 97/35966 by Minshull andStemmer, “Methods and Compositions for Cellular and MetabolicEngineering;” WO 99/41402 by Punnonen et al. “Targeting of GeneticVaccine Vectors;” WO 99/41383 by Punnonen et al. “Antigen LibraryImmunization;” WO 99/41369 by Punnonen et al. “Genetic Vaccine VectorEngineering;” WO 99/41368 by Punnonen et al. “Optimization ofImmunomodulatory Properties of Genetic Vaccines;” EP 752008 by Stemmerand Crameri, “DNA Mutagenesis by Random Fragmentation and Reassembly;”EP 0932670 by Stemmer “Evolving Cellular DNA Uptake by RecursiveSequence Recombination;” WO 99/23107 by Stemmer et al., “Modification ofVirus Tropism and Host Range by Viral Genome Shuffling;” WO 99/21979 byApt et al., “Human Papillomavirus Vectors;” WO 98/31837 by del Cardayreet al. “Evolution of Whole Cells and Organisms by Recursive SequenceRecombination;” WO 98/27230 by Patten and Stemmer, “Methods andCompositions for Polypeptide Engineering;” WO 98/13487 by Stemmer etal., “Methods for Optimization of Gene Therapy by Recursive SequenceShuffling and Selection;” WO 00/00632, “Methods for Generating HighlyDiverse Libraries;” WO 00/09679, “Methods for Obtaining in VitroRecombined Polynucleotide Sequence Banks and Resulting Sequences;” WO98/42832 by Arnold et al., “Recombination of Polynucleotide SequencesUsing Random or Defined Primers;” WO 99/29902 by Arnold et al., “Methodfor Creating Polynucleotide and Polypeptide Sequences;” WO 98/41653 byVind, “An in Vitro Method for Construction of a DNA Library;” WO98/41622 by Borchert et al., “Method for Constructing a Library UsingDNA Shuffling;” WO 98/42727 by Pati and Zarling, “Sequence Alterationsusing Homologous Recombination;” WO 00/18906 by Patten et al.,“Shuffling of Codon-Altered Genes;” WO 00/04190 by del Cardayre et al.“Evolution of Whole Cells and Organisms by Recursive Recombination;” WO00/42561 by Crameri et al., “Oligonucleotide Mediated Nucleic AcidRecombination;” WO 00/42559 by Selifonov and Stemmer “Methods ofPopulating Data Structures for Use in Evolutionary Simulations;” WO00/42560 by Selifonov et al., “Methods for Making Character Strings,Polynucleotides & Polypeptides Having Desired Characteristics;” WO01/23401 by Welch et al., “Use of Codon-Varied Oligonucleotide Synthesisfor Synthetic Shuffling;” and WO 01/64864 “Single-Stranded Nucleic AcidTemplate-Mediated Recombination and Nucleic Acid Fragment Isolation” byAffholter.

[0305] Certain U.S. applications provide additional details regardingvarious diversity generating methods, including “SHUFFLING OF CODONALTERED GENES” by Patten et al. filed Sep. 28, 1999, (U.S. Ser. No.09/407,800); “EVOLUTION OF WHOLE CELLS AND ORGANISMS BY RECURSIVESEQUENCE RECOMBINATION”, by del Cardayre et al. filed Jul. 15, 1998(USSN 09/166,188), and Jul. 15, 1999 (U.S. Pat. No. 6,379,964);“OLIGONUCLEOTIDE MEDIATED NUCLEIC ACID RECOMBINATION” by Crameri et al.,filed Sep. 28, 1999 (U.S. Pat. No. 6,376,246); “OLIGONUCLEOTIDE MEDIATEDNUCLEIC ACID RECOMBINATION” by Crameri et al., filed Jan. 18, 2000 (WO00/42561); “USE OF CODON-BASED OLIGONUCLEOTIDE SYNTHESIS FOR SYNTHETICSHUFFLING” by Welch et al., filed Sep. 28, 1999 (U.S. Pat. No.6,436,675); “METHODS FOR MAKING CHARACTER STRINGS, POLYNUCLEOTIDES &POLYPEPTIDES HAVING DESIRED CHARACTERISTICS” by Selifonov et al., filedJan. 18, 2000, (WO 00/42560); “METHODS FOR MAKING CHARACTER STRINGS,POLYNUCLEOTIDES & POLYPEPTIDES HAVING DESIRED CHARACTERISTICS” bySelifonov et al., filed Jul. 18, 2000 (U.S. Ser. No. 09/618,579);“METHODS OF POPULATING DATA STRUCTURES FOR USE IN EVOLUTIONARYSIMULATIONS” by Selifonov and Stemmer (WO 00/42559), filed Jan. 18,2000; and “SINGLE-STRANDED NUCLEIC ACID TEMPLATE-MEDIATED RECOMBINATIONAND NUCLEIC ACID FRAGMENT ISOLATION” by Affholter (U.S. Ser. No.60/186,482, filed Mar. 2, 2000).

[0306] In brief, several different general classes of sequencemodification methods, such as mutation, recombination, etc. areapplicable to the present invention and set forth in the referencesabove. That is, alterations to the component nucleic acid sequences toproduced modified gene fusion constructs can be performed by any numberof the protocols described, either before cojoining of the sequences, orafter the cojoining step. The following exemplify some of the differenttypes of preferred formats for diversity generation in the context ofthe present invention, including, e.g., certain recombination baseddiversity generation formats.

[0307] Nucleic acids can be recombined in vitro by any of a variety oftechniques discussed in the references above, including e.g., DNAsedigestion of nucleic acids to be recombined followed by ligation and/orPCR reassembly of the nucleic acids. For example, sexual PCR mutagenesiscan be used in which random (or pseudo random, or even non-random)fragmentation of the DNA molecule is followed by recombination, based onsequence similarity, between DNA molecules with different but relatedDNA sequences, in vitro, followed by fixation of the crossover byextension in a polymerase chain reaction. This process and many processvariants is described in several of the references above, e.g., inStemmer (1994) Proc. Natl. Acad. Sci. USA 91:10747-10751.

[0308] Similarly, nucleic acids can be recursively recombined in vivo,e.g., by allowing recombination to occur between nucleic acids in cells.Many such in vivo recombination formats are set forth in the referencesnoted above. Such formats optionally provide direct recombinationbetween nucleic acids of interest, or provide recombination betweenvectors, viruses, plasmids, etc., comprising the nucleic acids ofinterest, as well as other formats. Details regarding such proceduresare found in the references noted above.

[0309] Whole genome recombination methods can also be used in whichwhole genomes of cells or other organisms are recombined, optionallyincluding spiking of the genomic recombination mixtures with desiredlibrary components (e.g., genes corresponding to the pathways of thepresent invention). These methods have many applications, includingthose in which the identity of a target gene is not known. Details onsuch methods are found, e.g., in WO 98/31837 by del Cardayre et al.“Evolution of Whole Cells and Organisms by Recursive SequenceRecombination;” and in, e.g., WO 00/04190 by del Cardayre et al., alsoentitled “Evolution of Whole Cells and Organisms by Recursive SequenceRecombination.” Thus, any of these processes and techniques forrecombination, recursive recombination, and whole genome recombination,alone or in combination, can be used to generate the modified nucleicacid sequences and/or modified gene fusion constructs of the presentinvention.

[0310] Synthetic recombination methods can also be used, in whicholigonucleotides corresponding to targets of interest are synthesizedand reassembled in PCR or ligation reactions which includeoligonucleotides which correspond to more than one parental nucleicacid, thereby generating new recombined nucleic acids. Oligonucleotidescan be made by standard nucleotide addition methods, or can be made,e.g., by tri-nucleotide synthetic approaches. Details regarding suchapproaches are found in the references noted above, including, e.g., WO00/42561 by Crameri et al., “Oligonucleotide Mediated Nucleic AcidRecombination;” WO 01/23401 by Welch et al., “Use of Codon-VariedOligonucleotide Synthesis for Synthetic Shuffling;” WO 00/42560 bySelifonov et al., “Methods for Making Character Strings, Polynucleotidesand Polypeptides Having Desired Characteristics;” and WO 00/42559 bySelifonov and Stemmer “Methods of Populating Data Structures for Use inEvolutionary Simulations.”

[0311] In silico methods of recombination can be effected in whichgenetic algorithms are used in a computer to recombine sequence stringswhich correspond to homologous (or even non-homologous) nucleic acids.The resulting recombined sequence strings are optionally converted intonucleic acids by synthesis of nucleic acids which correspond to therecombined sequences, e.g., in concert with oligonucleotide synthesisgene reassembly techniques. This approach can generate random, partiallyrandom or designed variants. Many details regarding in silicorecombination, including the use of genetic algorithms, geneticoperators and the like in computer systems, combined with generation ofcorresponding nucleic acids (and/or proteins), as well as combinationsof designed nucleic acids and/or proteins (e.g., based on cross-oversite selection) as well as designed, pseudo-random or randomrecombination methods are described in WO 00/42560 by Selifonov et al.,“Methods for Making Character Strings, Polynucleotides and PolypeptidesHaving Desired Characteristics” and WO 00/42559 by Selifonov and Stemmer“Methods of Populating Data Structures for Use in EvolutionarySimulations.” Extensive details regarding in silico recombinationmethods are found in these applications. This methodology is generallyapplicable to the present invention in providing for recombination ofnucleic acid sequences and/or gene fusion constructs encoding proteinsinvolved in various metabolic pathways (such as, for example, carotenoidbiosynthetic pathways, ectoine biosynthetic pathways,polyhydroxyalkanoate biosynthetic pathways, aromatic polyketidebiosynthetic pathways, and the like) in silico and/or the generation ofcorresponding nucleic acids or proteins.

[0312] Many methods of accessing natural diversity, e.g., byhybridization of diverse nucleic acids or nucleic acid fragments tosingle-stranded templates, followed by polymerization and/or ligation toregenerate full-length sequences, optionally followed by degradation ofthe templates and recovery of the resulting modified nucleic acids canbe similarly used. In one method employing a single-stranded template,the fragment population derived from the genomic library(ies) isannealed with partial, or, often approximately full length ssDNA or RNAcorresponding to the opposite strand. Assembly of complex chimeric genesfrom this population is then mediated by nuclease-base removal ofnon-hybridizing fragment ends, polymerization to fill gaps between suchfragments and subsequent single stranded ligation. The parentalpolynucleotide strand can be removed by digestion (e.g., if RNA oruracil-containing), magnetic separation under denaturing conditions (iflabeled in a manner conducive to such separation) and other availableseparation/purification methods. Alternatively, the parental strand isoptionally co-purified with the chimeric strands and removed duringsubsequent screening and processing steps. Additional details regardingthis approach are found, e.g., in “Single-Stranded Nucleic AcidTemplate-Mediated Recombination and Nucleic Acid Fragment Isolation” byAffholter, WO 01/64864.

[0313] In another approach, single-stranded molecules are converted todouble-stranded DNA (dsDNA) and the dsDNA molecules are bound to a solidsupport by ligand-mediated binding. After separation of unbound DNA, theselected DNA molecules are released from the support and introduced intoa suitable host cell to generate a library of enriched sequences whichhybridize to the probe. A library produced in this manner provides adesirable substrate for further diversification using any of theprocedures described herein.

[0314] Any of the preceding general recombination formats can bepracticed in a reiterative fashion (e.g., one or more cycles ofmutation/recombination or other diversity generation methods, optionallyfollowed by one or more selection methods) to generate a more diverseset of recombinant nucleic acids.

[0315] Mutagenesis employing polynucleotide chain termination methodshave also been proposed (see e.g., U.S. Pat. No. 5,965,408, “Method ofDNA reassembly by interrupting synthesis” to Short, and the referencesabove), and can be applied to the present invention. In this approach,double stranded DNAs corresponding to one or more genes sharing regionsof sequence similarity are combined and denatured, in the presence orabsence of primers specific for the gene. The single strandedpolynucleotides are then annealed and incubated in the presence of apolymerase and a chain terminating reagent (e.g., ultraviolet, gamma orX-ray irradiation; ethidium bromide or other intercalators; DNA bindingproteins, such as single strand binding proteins, transcriptionactivating factors, or histones; polycyclic aromatic hydrocarbons;trivalent chromium or a trivalent chromium salt; or abbreviatedpolymerization mediated by rapid thermocycling; and the like), resultingin the production of partial duplex molecules. The partial duplexmolecules, e.g., containing partially extended chains, are thendenatured and reannealed in subsequent rounds of replication or partialreplication resulting in polynucleotides which share varying degrees ofsequence similarity and which are diversified with respect to thestarting population of DNA molecules. Optionally, the products, orpartial pools of the products, can be amplified at one or more stages inthe process. Polynucleotides produced by a chain termination method,such as described above, are suitable substrates for any other describedrecombination format.

[0316] Diversity also can be generated in nucleic acids or populationsof nucleic acids using a recombinational procedure termed “incrementaltruncation for the creation of hybrid enzymes” (“ITCHY”) described inOstermeier et al. (1999) “A combinatorial approach to hybrid enzymesindependent of DNA homology” Nature Biotech 17:1205. This approach canbe used to generate an initial library of variants which can optionallyserve as a substrate for one or more in vitro or in vivo recombinationmethods. See, also, Ostermeier et al. (1999) “Combinatorial ProteinEngineering by Incremental Truncation,” Proc. Natl. Acad. Sci. USA, 96:3562-67; and Ostermeier et al. (1999), “Incremental Truncation as aStrategy in the Engineering of Novel Biocatalysts,” Biological andMedicinal Chemistry, 7: 2139-44.

[0317] Mutational methods which result in the alteration of individualnucleotides or groups of contiguous or non-contiguous nucleotides can befavorably employed to introduce nucleotide diversity into the nucleicacid sequences and/or gene fusion constructs of the present invention.Many mutagenesis methods are found in the above-cited references;additional details regarding mutagenesis methods can be found infollowing, which can also be applied to the present invention.

[0318] For example, error-prone PCR can be used to generate nucleic acidvariants. Using this technique, PCR is performed under conditions wherethe copying fidelity of the DNA polymerase is low, such that a high rateof point mutations is obtained along the entire length of the PCRproduct. Examples of such techniques are found in the references aboveand, e.g., in Leung et al. (1989) Technique 1:11-15 and Caldwell et al.(1992) PCR Methods Applic. 2:28-33. Similarly, assembly PCR can be used,in a process which involves the assembly of a PCR product from a mixtureof small DNA fragments. A large number of different PCR reactions canoccur in parallel in the same reaction mixture, with the products of onereaction priming the products of another reaction.

[0319] Oligonucleotide directed mutagenesis can be used to introducesite-specific mutations in a nucleic acid sequence of interest. Examplesof such techniques are found in the references above and, e.g., inReidhaar-Olson et al. (1988) Science, 241:53-57. Similarly, cassettemutagenesis can be used in a process that replaces a small region of adouble stranded DNA molecule with a synthetic oligonucleotide cassettethat differs from the native sequence. The oligonucleotide can contain,e.g., completely and/or partially randomized native sequence(s).

[0320] Recursive ensemble mutagenesis is a process in which an algorithmfor protein mutagenesis is used to produce diverse populations ofphenotypically related mutants, members of which differ in amino acidsequence. This method uses a feedback mechanism to monitor successiverounds of combinatorial cassette mutagenesis. Examples of this approachare found in Arkin & Youvan (1992) Proc. Natl. Acad. Sci. USA89:7811-7815.

[0321] Exponential ensemble mutagenesis can be used for generatingcombinatorial libraries with a high percentage of unique and functionalmutants. Small groups of residues in a sequence of interest arerandomized in parallel to identify, at each altered position, aminoacids which lead to functional proteins. Examples of such procedures arefound in Delegrave & Youvan (1993) Biotechnology Research 11:1548-1552.

[0322] In vivo mutagenesis can be used to generate random mutations inany cloned DNA of interest by propagating the DNA, e.g., in a strain ofE. coli that carries mutations in one or more of the DNA repairpathways. These “mutator” strains have a higher random mutation ratethan that of a wild-type parent. Propagating the DNA in one of thesestrains will eventually generate random mutations within the DNA. Suchprocedures are described in the references noted above.

[0323] Other procedures for introducing diversity into a genome, e.g. abacterial, fungal, animal or plant genome can be used in conjunctionwith the above described and/or referenced methods. For example, inaddition to the methods above, techniques have been proposed whichproduce nucleic acid multimers suitable for transformation into avariety of species (see, e.g., Schellenberger U.S. Pat. No. 5,756,316and the references above). Transformation of a suitable host with suchmultimers, consisting of genes that are divergent with respect to oneanother, (e.g., derived from natural diversity or through application ofsite directed mutagenesis, error prone PCR, passage through mutagenicbacterial strains, and the like), provides a source of nucleic aciddiversity for DNA diversification, e.g., by an in vivo recombinationprocess as indicated above.

[0324] Alternatively, a multiplicity of monomeric polynucleotidessharing regions of partial sequence similarity can be transformed into ahost species and recombined in vivo by the host cell. Subsequent roundsof cell division can be used to generate libraries, members of which,include a single, homogenous population, or pool of monomericpolynucleotides. Alternatively, the monomeric nucleic acids can berecovered by standard techniques, e.g., PCR and/or cloning, andrecombined in any of the recombination formats, including recursiverecombination formats, described above.

[0325] Methods for generating multispecies expression libraries havebeen described (in addition to the references noted above, see, e.g.,Peterson et al. (1998) U.S. Pat. No. 5,783,431 “METHODS FOR GENERATINGAND SCREENING NOVEL METABOLIC PATHWAYS;” and Thompson, et al. (1998)U.S. Pat. No. 5,824,485 METHODS FOR GENERATING AND SCREENING NOVELMETABOLIC PATHWAYS) and their use to identify protein activities ofinterest has been proposed (in addition to the references noted above,see, Short (1999) U.S. Pat. No. 5,958,672 “PROTEIN ACTIVITY SCREENING OFCLONES HAVING DNA FROM UNCULTIVATED MICROORGANISMS”). Multispeciesexpression libraries include, in general, libraries comprising cDNA orgenomic sequences from a plurality of species or strains, operablylinked to appropriate regulatory sequences, in an expression cassette.The cDNA and/or genomic sequences are optionally randomly ligated tofurther enhance diversity. The vector can be a shuttle vector suitablefor transformation and expression in more than one species of hostorganism, e.g., bacterial species or eukaryotic cells. In some cases,the library is biased by preselecting sequences which encode a proteinof interest, or which hybridize to a nucleic acid of interest. Any suchlibraries can be provided as substrates for any of the methods hereindescribed.

[0326] The above described procedures have been largely directed toincreasing nucleic acid and/or encoded protein diversity. However, inmany cases, not all of the diversity is useful, e.g., functional, andcontributes merely to increasing the background of variants that must bescreened or selected to identify the few favorable variants. In someapplications, it is desirable to preselect or prescreen libraries (e.g.,an amplified library, a genomic library, a cDNA library, a normalizedlibrary, etc.) or other substrate nucleic acids prior todiversification, e.g., by recombination-based mutagenesis procedures, orto otherwise bias the substrates towards nucleic acids that encodefunctional products. For example, in the case of antibody engineering,it is possible to bias the diversity generating process towardantibodies with functional antigen binding sites by taking advantage ofin vivo recombination events prior to manipulation by any of thedescribed methods. For example, recombined CDRs derived from B cell cDNAlibraries can be amplified and assembled into framework regions (e.g.,Jirholt et al. (1998) “Exploiting sequence space: shuffling in vivoformed complementarity determining regions into a master framework” Gene215: 471) prior to diversifying according to any of the methodsdescribed herein.

[0327] Libraries can be biased towards nucleic acids which encodeproteins with desirable enzyme activities. For example, afteridentifying a clone from a library which exhibits a specified activity,the clone can be mutagenized using any known method for introducing DNAalterations. A library comprising the mutagenized homologues is thenscreened for a desired activity, which can be the same as or differentfrom the initially specified activity. An example of such a procedure isproposed in Short (1999) U.S. Pat. No. 5,939,250 for “PRODUCTION OFENZYMES HAVING DESIRED ACTIVITIES BY MUTAGENESIS.” Desired activitiescan be identified by any method known in the art. For example, WO99/10539 proposes that gene libraries can be screened by combiningextracts from the gene library with components obtained frommetabolically rich cells and identifying combinations which exhibit thedesired activity. It has also been proposed (e.g., WO 98/58085) thatclones with desired activities can be identified by inserting bioactivesubstrates into samples of the library, and detecting bioactivefluorescence corresponding to the product of a desired activity using afluorescent analyzer, e.g., a flow cytometry device, a CCD, afluorometer, or a spectrophotometer.

[0328] Libraries can also be biased towards nucleic acids which havespecified characteristics, e.g., hybridization to a selected nucleicacid probe. For example, WO 99/10539 proposes that polynucleotidesencoding a desired activity (e.g., an enzymatic activity, for example: alipase, an esterase, a protease, a glycosidase, a glycosyl transferase,a phosphatase, a kinase, an oxygenase, a peroxidase, a hydrolase, ahydratase, a nitrilase, a transaminase, an amidase or an acylase) can beidentified from among genomic DNA sequences. In particular, singlestranded DNA molecules from a population of genomic DNA are hybridizedto a ligand-conjugated probe. The genomic DNA can be derived from eithera cultivated or uncultivated microorganism, or from an environmentalsample. Alternatively, the genomic DNA can be derived from amulticellular organism, or a tissue derived therefrom. Second strandsynthesis can be conducted directly from the hybridization probe used inthe capture, with or without prior release from the capture medium or bya wide variety of other strategies known in the art. Alternatively, theisolated single-stranded genomic DNA population can be fragmentedwithout further cloning and used directly in, e.g., arecombination-based approach, that employs a single-stranded template,as described above.

[0329] “Non-stochastic” methods of generating nucleic acids andpolypeptides are described in Short “Non-Stochastic Generation ofGenetic Vaccines and Enzymes” WO 00/46344. These methods, includingproposed non-stochastic polynucleotide reassembly and site-saturationmutagenesis methods can be applied to the present invention as well.Random or semi-random mutagenesis using doped or degenerateoligonucleotides is also described in, e.g., Arkin and Youvan (1992)“Optimizing nucleotide mixtures to encode specific subsets of aminoacids for semi-random mutagenesis” Biotechnology 10:297-300;Reidhaar-Olson et al. (1991) “Random mutagenesis of protein sequencesusing oligonucleotide cassettes” Methods Enzymol. 208:564-86; Lim andSauer (1991) “The role of internal packing interactions in determiningthe structure and stability of a protein” J. Mol. Biol. 219:359-76;Breyer and Sauer (1989) “Mutational analysis of the fine specificity ofbinding of monoclonal antibody 51F to lambda repressor” J. Biol. Chem.264:13355-60); “Walk-Through Mutagenesis” (Crea, R; U.S. Pat. Nos.5,830,650 and 5,798,208, and EP Patent 0527809 B1.

[0330] It will readily be appreciated that any of the above describedtechniques suitable for enriching a library prior to diversification canalso be used to screen the products, or libraries of products, producedby the diversity generating methods. Any of the above described methodscan be practiced recursively or in combination to alter nucleic acids,e.g., GAT encoding polynucleotides.

[0331] Kits for mutagenesis, library construction and other diversitygeneration methods are also commercially available. For example, kitsare available from, e.g., Stratagene (e.g., QuickChange™ site-directedmutagenesis kit; and Chameleon™ double-stranded, site-directedmutagenesis kit); Bio/Can Scientific, Bio-Rad (e.g., using the Kunkelmethod described above); Boehringer Mannheim Corp.; ClonetechLaboratories; DNA Technologies; Epicentre Technologies (e.g., 5 prime 3prime kit); Genpak Inc.; Lemargo Inc.; Life Technologies (Gibco BRL);New England Biolabs; Pharmacia Biotech; Promega Corp.; QuantumBiotechnologies; Amersham International plc (e.g., using the Ecksteinmethod above); and Anglian Biotechnology Ltd (e.g., using theCarter/Winter method above).

[0332] The above references provide many mutational formats, includingrecombination, recursive recombination, recursive mutation andcombinations of recombination with other forms of mutagenesis, as wellas many modifications of these formats. Regardless of the diversitygeneration format that is used, the nucleic acids of the presentinvention can be recombined (with each other, or with related (or evenunrelated) sequences) to produce a diverse set of recombinant nucleicacids for use in the gene fusion constructs and modified gene fusionconstructs of the present invention, including, e.g., sets of homologousnucleic acids, as well as corresponding polypeptides.

[0333] Many of the above-described methodologies for generating modifiedpolynucleotides generate a large number of diverse variants of aparental sequence or sequences. In some preferred embodiments of theinvention the modification technique (e.g., some form of shuffling) isused to generate a library of variants that is then screened for amodified polynucleotide or pool of modified polynucleotides encodingsome desired functional attribute, e.g., improved GAT activity.Exemplary enzymatic activities that can be screened for includecatalytic rates (conventionally characterized in terms of kineticconstants such as k_(cat) and K_(M)), substrate specificity, andsusceptibility to activation or inhibition by substrate, product orother molecules (e.g., inhibitors or activators).

[0334] One example of selection for a desired enzymatic activity entailsgrowing host cells under conditions that inhibit the growth and/orsurvival of cells that do not sufficiently express an enzymatic activityof interest, e.g. the GAT activity. Using such a selection process caneliminate from consideration all modified polynucleotides except thoseencoding a desired enzymatic activity. For example, in some embodimentsof the invention host cells are maintained under conditions that inhibitcell growth or survival in the absence of sufficient levels of GAT,e.g., a concentration of glyphosate that is lethal or inhibits thegrowth of a wild-type plant of the same variety that either lacks ordoes not express a GAT polynucleotide. Under these conditions, only ahost cell harboring a modified nucleic acid that encodes enzymaticactivity or activities able to catalyze production of sufficient levelsof the product will survive and grow. Some embodiments of the inventionemploy multiple rounds of screening at increasing concentrations ofglyphosate or a glyphosate analog.

[0335] In some embodiments of the invention, mass spectrometry is usedto detect the acetylation of glyphosate, or a glyphosate analog ormetabolite. The use of mass spectrometry is described in more detail inthe Examples below.

[0336] For convenience and high throughput it will often be desirable toscreen/select for desired modified nucleic acids in a microorganism,e.g., a bacteria such as E. coli. On the other hand, screening in plantcells or plants can in some cases be preferable where the ultimate aimis to generate a modified nucleic acid for expression in a plant system.

[0337] In some preferred embodiments of the invention throughput isincreased by screening pools of host cells expressing different modifiednucleic acids, either alone or as part of a gene fusion construct. Anypools showing significant activity can be deconvoluted to identifysingle clones expressing the desirable activity.

[0338] The skilled artisan will recognize that the relevant assay,screening or selection method will vary depending upon the desired hostorganism and other parameters known in the art. It is normallyadvantageous to employ an assay that can be practiced in ahigh-throughput format.

[0339] In high-throughput assays, it is possible to screen up to severalthousand different variants in a single day. For example, each well of amicrotiter plate can be used to run a separate assay, or, ifconcentration or incubation time effects are to be observed, every 5-10wells can test a single variant.

[0340] In addition to fluidic approaches, it is possible, as mentionedabove, simply to grow cells on media plates that select for the desiredenzymatic or metabolic function. This approach offers a simple andhigh-throughput screening method.

[0341] A number of well known robotic systems have also been developedfor solution phase chemistries useful in assay systems. These systemsinclude automated workstations like the automated synthesis apparatusdeveloped by Takeda Chemical Industries, LTD. (Osaka, Japan) and manyrobotic systems utilizing robotic arms (Zymate II, Zymark Corporation,Hopkinton, Mass.; and Orca, Hewlett-Packard, Palo Alto, Calif.) whichmimic the manual synthetic operations performed by a scientist. Any ofthe above devices are suitable for application to the present invention.The nature and implementation of modifications to these devices (if any)so that they can operate as discussed herein with reference to theintegrated system will be apparent to persons skilled in the relevantart.

[0342] High-throughput screening systems are commercially available(see, e.g., Zymark Corp., Hopkinton, Mass.; Air Technical Industries,Mentor, Ohio; Beckman Instruments, Inc. Fullerton, Calif.; PrecisionSystems, Inc., Natick, Mass., etc.). These systems typically automateentire procedures including all sample and reagent pipetting, liquiddispensing, timed incubations, and final readings of the microplate indetector(s) appropriate for the particular assay. These configurablesystems provide high throughput and rapid start up as well as a highdegree of flexibility and customization.

[0343] The manufacturers of such systems provide detailed protocols forthe various high throughput devices. Thus, for example, Zymark Corp.provides technical bulletins describing screening systems for detectingthe modulation of gene transcription, ligand binding, and the like.Microfluidic approaches to reagent manipulation have also beendeveloped, e.g., by Caliper Technologies (Mountain View, Calif.).

[0344] Optical images viewed (and, optionally, recorded) by a camera orother recording device (e.g., a photodiode and data storage device) areoptionally further processed in any of the embodiments herein, e.g., bydigitizing the image and/or storing and analyzing the image on acomputer. A variety of commercially available peripheral equipment andsoftware is available for digitizing, storing and analyzing a digitizedvideo or digitized optical image, e.g., using PC (Intel ×86 or Pentiumchip compatible DOS™, OS™ WINDOWS™, WINDOWS NT™ or WINDOWS 95™ basedmachines), MACINTOSH™, or UNIX based (e.g., SUN™ work station)computers.

[0345] One conventional system carries light from the assay device to a.cooled charge-coupled device (CCD) camera, a common use in the art. ACCD camera includes an array of picture elements (pixels). The lightfrom the specimen is imaged on the CCD. Particular pixels correspondingto regions of the specimen (e.g., individual hybridization sites on anarray of biological polymers) are sampled to obtain light intensityreadings for each position. Multiple pixels are processed in parallel toincrease speed. The apparatus and methods of the invention are easilyused for viewing any sample, e.g. by fluorescent or dark fieldmicroscopic techniques.

[0346] Other Polynucleotide Compositions

[0347] The invention also includes compositions comprising two or morepolynucleotides of the invention (e.g., as substrates forrecombination). The composition can comprise a library of recombinantnucleic acids, where the library contains at least 2, 3, 5, 10, 20, or50 or more polynucleotides. The polynucleotides are optionally clonedinto expression vectors, providing expression libraries.

[0348] The invention also includes compositions produced by digestingone or more polynucleotide of the invention with a restrictionendonuclease, an RNAse, or a DNAse (e.g., as is performed in certain ofthe recombination formats noted above); and compositions produced byfragmenting or shearing one or more polynucleotide of the invention bymechanical means (e.g., sonication, vortexing, and the like), which canalso be used to provide substrates for recombination in the methodsabove. Similarly, compositions comprising sets of oligonucleotidescorresponding to more than one nucleic acid of the invention are usefulas recombination substrates and are a feature of the invention. Forconvenience, these fragmented, sheared, or oligonucleotide synthesizedmixtures are referred to as fragmented nucleic acid sets.

[0349] Also included in the invention are compositions produced byincubating one or more of the fragmented nucleic acid sets in thepresence of ribonucleotide- or deoxyribonucelotide triphosphates and anucleic acid polymerase. This resulting composition forms arecombination mixture for many of the recombination formats noted above.The nucleic acid polymerase may be an RNA polymerase, a DNA polymerase,or an RNA-directed DNA polymerase (e.g., a “reverse transcriptase”); thepolymerase can be, e.g., a thermostable DNA polymerase (such as, VENT,TAQ, or the like).

[0350] Integrated Systems

[0351] The present invention provides computers, computer readable mediaand integrated systems comprising character strings corresponding to thesequence information herein for the polypeptides and nucleic acidsherein, including, e.g., those sequences listed herein and the varioussilent substitutions and conservative substitutions thereof.

[0352] For example, various methods and genetic algorithms (GAs) knownin the art can be used to detect homology or similarity betweendifferent character strings, or can be used to perform other desirablefunctions such as to control output files, provide the basis for makingpresentations of information including the sequences and the like.Examples include BLAST, discussed supra.

[0353] Thus, different types of homology and similarity of variousstringency and length can be detected and recognized in the integratedsystems described herein. For example, many homology determinationmethods have been designed for comparative analysis of sequences ofbiopolymers, for spell-checking in word processing, and for dataretrieval from various databases. With an understanding of double-helixpair-wise complement interactions among 4 principal nucleobases innatural polynucleotides, models that simulate annealing of complementaryhomologous polynucleotide strings can also be used as a foundation ofsequence alignment or other operations typically performed on thecharacter strings corresponding to the sequences herein (e.g.,word-processing manipulations, construction of figures comprisingsequence or subsequence character strings, output tables, etc.). Anexample of a software package with GAs for calculating sequencesimilarity is BLAST, which can be adapted to the present invention byinputting character strings corresponding to the sequences herein.

[0354] Similarly, standard desktop applications such as word processingsoftware (e.g., Microsoft Word™ or Corel WordPerfect™) and databasesoftware (e.g., spreadsheet software such as Microsoft Excel™, CorelQuattro Pro™, or database programs such as Microsoft Access™ orParadox™) can be adapted to the present invention by inputting acharacter string corresponding to the GAT homologues of the invention(either nucleic acids or proteins, or both). For example, the integratedsystems can include the foregoing software having the appropriatecharacter string information, e.g., used in conjunction with a userinterface (e.g., a GUI in a standard operating system such as a Windows,Macintosh or LINUX system) to manipulate strings of characters. Asnoted, specialized alignment programs such as BLAST can also beincorporated into the systems of the invention for alignment of nucleicacids or proteins (or corresponding character strings).

[0355] Integrated systems for analysis in the present inventiontypically include a digital computer with GA software for aligningsequences, as well as data sets entered into the software systemcomprising any of the sequences herein. The computer can be, e.g., a PC(Intel ×86 or Pentium chip compatible DOS™, OS2™ WINDOWS™ WINDOWS NT™,WINDOWS95™, WINDOWS98™ LINUX based machine, a MACINTOSH™, Power PC, or aUNIX based (e.g., SUN™ work station) machine) or other commerciallycommon computer which is known to one of skill. Software for aligning orotherwise manipulating sequences is available, or can easily beconstructed by one of skill using a standard programming language suchas Visualbasic, Fortran, Basic, Java, or the like.

[0356] Any controller or computer optionally includes a monitor which isoften a cathode ray tube (“CRT”) display, a flat panel display (e.g.,active matrix liquid crystal display, liquid crystal display), orothers. Computer circuitry is often placed in a box which includesnumerous integrated circuit chips, such as a microprocessor, memory,interface circuits, and others. The box also optionally includes a harddisk drive, a floppy disk drive, a high capacity removable drive such asa writeable CD-ROM, and other common peripheral elements. Inputtingdevices such as a keyboard or mouse optionally provide for input from auser and for user selection of sequences to be compared or otherwisemanipulated in the relevant computer system.

[0357] The computer typically includes appropriate software forreceiving user instructions, either in the form of user input into setparameter fields, e.g., in a GUI, or in the form of preprogrammedinstructions, e.g., preprogrammed for a variety of different specificoperations. The software then converts these instructions to appropriatelanguage for instructing the operation of the fluid direction andtransport controller to carry out the desired operation.

[0358] The software can also include output elements for controllingnucleic acid synthesis (e.g., based upon a sequence or an alignment of asequences herein) or other operations which occur downstream from analignment or other operation performed using a character stringcorresponding to a sequence herein. Nucleic acid synthesis equipmentcan, accordingly, be a component in one or more integrated systemsherein.

[0359] In an additional aspect, the present invention provides kitsembodying the methods, composition, systems and apparatus herein. Kitsof the invention optionally comprise one or more of the following: (1)an apparatus, system, system component or apparatus component asdescribed herein; (2) instructions for practicing the methods describedherein, and/or for operating the apparatus or apparatus componentsherein and/or for using the compositions herein; (3) one or more GATcompositions or components; (4) a container for holding components orcompositions, and, (5) packaging materials.

[0360] In a further aspect, the present invention provides for the useof any apparatus, apparatus component, composition or kit herein, forthe practice of any method or assay herein, and/or for the use of anyapparatus or kit to practice any assay or method herein.

[0361] Host Cells and Organisms

[0362] The host cell can be eukaryotic, for example, a eukaryotic cell,a plant cell, an animal cell, a protoplast, or a tissue culture cell.The host cell optionally comprises a plurality of cells, for example, anorganism. Alternatively, the host cell can be prokaryotic including, butnot limited to, bacteria (i.e., gram positive bacteria, purple bacteria,green sulfur bacteria, green non-sulfur bacteria, cyanobacteria,spirochetes, thermatogales, flavobacteria, and bacteroides) andarchaebacteria (i.e., Korarchaeota, Thermoproteus, Pyrodictium,Thermococcales, Methanogens, Archaeoglobus, and extreme Halophiles).

[0363] Transgenic plants, or plant cells, incorporating the GAT nucleicacids, and/or expressing the GAT polypeptides of the invention are afeature of the invention. The transformation of plant cells andprotoplasts can be carried out in essentially any of the various waysknown to those skilled in the art of plant molecular biology, including,but not limited to, the methods described herein. See, in general,Methods in Enzymology Vol. 153 (Recombinant DNA Part D) Wu and Grossman(eds.) 1987, Academic Press; and Weising et al., Ann. Rev. Genet. 22:421-477 (1988), incorporated herein by reference. For example, the DNAconstruct may be introduced directly into the genomic DNA of the plantcell using techniques such as electroporation, PEG-mediatedtransfection, particle bombardment, silicon fiber delivery, ormicroinjection of plant cell protoplasts or embryogenic callus. See,e.g., Tomes, et al., Direct DNA Transfer into Intact Plant Cells ViaMicroprojectile Bombardment. pp.197-213 in Plant Cell, Tissue and OrganCulture, Fundamental Methods, eds. O. L. Gamborg and G. C. Phillips,Springer-Verlag Berlin, Heidelberg, N.Y., 1995. Further methods fortransforming various host cells are disclosed in Klein et al.“Transformation of microbes, plants and animals by particlebombardment”, Bio/Technol., New York, N.Y., Nature Publishing Company,March 1992, v. 10 (3) pp. 286-291.

[0364] The introduction of DNA constructs using polyethylene glycolprecipitation is described in Paszkowski et al., Embo J. 3:2717-2722(1984). Electroporation techniques are described in Fromm et al., Proc.Natl. Acad. Sci. 82:5824 (1985). Ballistic transformation techniques aredescribed in Klein et al., Nature 327: 70-73 (1987).

[0365] Alternatively, the DNA constructs may be combined with suitableT-DNA flanking regions and introduced into a conventional Agrobacteriumtumefaciens host vector. The virulence functions of the Agrobacteriumtumefaciens host will direct the insertion of the construct and adjacentmarker into the plant cell DNA when the cell is infected by thebacteria. See, U.S. Pat. No. 5,591,616.

[0366]Agrobacterium tumefaciens-meditated transformation techniques arewell described in the scientific literature. See, for example Horsch etal., Science 233:496-498 (1984), and Fraley et al., Proc. Natl. Acad.Sci. 80:4803 (1983). For instance, Agrobacterium transformation of maizeis described in U.S. Pat. Nos. 5,550,318 and 5,981,840.

[0367] Other methods of transformation include (1) Agrobacteriumrhizogenes-mediated transformation (see, e.g., Lichtenstein and FullerIn: Genetic Engineering, Vol. 6, P W J Rigby, ed., London, AcademicPress, 1987; Lichtenstein, C. P., and Draper, J,. In: DNA Cloning, Vol.II, D. M. Glover, Ed., Oxford, IRI Press, 1985; WO 88/02405 describesthe use of A. rhizogenes strain A4 and its Ri plasmid along with A.tumefaciens vectors pARC8 or pARC16); (2) liposome-mediated DNA uptake(see, e.g., Freeman et al., Plant Cell Physiol. 25:1353, 1984; (3) thevortexing method (see, e.g., Kindle, Proc. Natl. Acad. Sci., USA87:1228, (1990).

[0368] DNA can also be introduced into plants by direct DNA transferinto pollen as described by Zhou et al., Methods in Enzymology, 101:433(1983); D. Hess, Intern Rev. Cytol., 107:367 (1987); and Luo et al.,Plane Mol. Biol. Reporter, 6:165 (1988). Expression of polypeptidecoding nucleic acids can be obtained by injection of the DNA intoreproductive organs of a plant as described by Pena et al., Nature,325:274 (1987). DNA can also be injected directly into the cells ofimmature embryos and the rehydration of desiccated embryos as describedby Neuhaus et al., Theor. Appl. Genet., 75:30 (1987); and Benbrook etal., in Proceedings Bio Expo 1986, Butterworth, Stoneham, Mass., pp.27-54 (1986).

[0369] Animal and lower eukaryotic (e.g., yeast) host cells arecompetent or rendered competent for transfection by various means. Thereare several well-known methods of introducing DNA into animal cells.These methods include: calcium phosphate precipitation; fusion of therecipient cells with bacterial protoplasts containing the DNA; treatmentof the recipient cells with liposomes containing the DNA; DEAE dextran;electroporation; biolistics; and micro-injection of the DNA directlyinto the cells. The transfected cells are cultured by means well knownin the art. See, Kuchler, R. J., Biochemical Methods in Cell Culture andVirology, Dowden, Hutchinson and Ross, Inc. (1977). As used herein, theterm “transformation” means alteration of the genotype of a host plantby the introduction of a nucleic acid sequence, e.g., a “heterologous”or “foreign” nucleic acid sequence. The heterologous nucleic acidsequence need not necessarily originate from a different source but itwill, at some point, have been external to the cell into which isintroduced.

[0370] In addition to Berger, Ausubel and Sambrook, useful generalreferences for plant cell cloning, culture and regeneration includeJones (ed.) (1995) Plant Gene Transfer and Expression Protocols—Methodsin Molecular Biology, Volume 49 Humana Press Towata N.J.; Payne et al.(1992) Plant Cell and Tissue Culture in Liquid Systems John Wiley &Sons, Inc. New York, N.Y. (“Payne”); and Gamborg and Phillips (eds.)(1995) Plant Cell Tissue and Organ Culture; Fundamental Methods SpringerLab Manual, Springer-Verlag (Berlin, Heidelberg, N.Y.) (“Gamborg”). Avariety of cell culture media are described in Atlas and Parks (eds.)The Handbook of Microbiological Media (1993) CRC Press, Boca Raton, Fla.(“Atlas”). Additional information for plant cell culture is found inavailable commercial literature such as the Life Science Research CellCulture Catalogue (1998) from Sigma-Aldrich, Inc. (St Louis, Mo.)(Sigma-LSRCCC) and, e.g., the Plant Culture Catalogue and supplement(1997) also from Sigma-Aldrich, Inc. (St Louis, Mo.) (Sigma-PCCS).Additional details regarding plant cell culture are found in Croy, (ed.)(1993) Plant Molecular Biology Bios Scientific Publishers, Oxford, U.K.

[0371] In an embodiment of this invention, recombinant vectors includingone or more GAT polynucleotides, suitable for the transformation ofplant cells are prepared. A DNA sequence encoding for the desired GATpolypeptide, e.g., selected from among SEQ ID NO: 1-5, 11-262, 516-567,620, 622, 624, 626, 628, 630, 632, 634, 636, 638, 640, 642, 644, 646,648, 650, 652, 654, 656, 658, 660, 662, 664, 666, 668, 670, 672, 674,676, 678, 680, 682, 684, 686, 688, 690, 692, 694, 696, 698, 700, 702,704, 706, 708, 710, 712, 714, 716, 718, 720, 722, 724, 726, 728, 730,732, 734, 736, 738, 740, 742, 744, 746, 748, 750, 752, 754, 756, 758,760, 762, 764, 766, 768, 770, 772, 774, 776, 778, 780, 782, 784, 786,788, 790, 792, 794, 796, 798, 800, 802, 804, 806, 808, 810, and 812, isconveniently used to construct a recombinant expression cassette whichcan be introduced into the desired plant. In the context of the presentinvention, an expression cassette will typically comprise a selected GATpolynucleotide operably linked to a promoter sequence and othertranscriptional and translational initiation regulatory sequences whichare sufficient to direct the transcription of the GAT sequence in theintended tissues (e.g., entire plant, leaves, roots, etc.) of thetransformed plant.

[0372] A number of promoters can be used in the practice of the presentinvention. The promoters can be selected based on the desired outcome.That is, the nucleic acids can be combined with constitutive,tissue-preferred, or other promoters for expression in plants.

[0373] Constitutive promoters include, for example, the core promoter ofthe Rsyn7 promoter and other constitutive promoters disclosed in WO99/43838 and U.S. Pat. No. 6,072,050; the core CaMV 35S promoter (Odellet al (1985) Nature 313:810-812); rice actin (McElroy et al. (1990)Plant Cell 2:163-171); ubiquitin (Christensen et al (1989) Plant Mol.Biol. 12:619-632 and Christensen et al. (1992) Plant Mol Biol.18:675-689); pEMU (Last et al. (1991) Theor. Appl. Genet. 81:581-588);MAS (Velten et al. (1984) EMBO J. 3:2723-2730); ALS promoter (U.S. Pat.No. 5,659,026), and the like. Other constitutive promoters include, forexample, those disclosed in U.S. Pat. Nos. 5,608,149; 5,608,144;5,604,121; 5,569,597; 5,466,785; 5,399,680; 5,268,463; 5,608,142; and6,177,611.

[0374] Chemical-regulated promoters can be used to modulate theexpression of a gene in a plant through the application of an exogenouschemical regulator. Depending upon the objective, the promoter may be achemical-inducible promoter, where application of the chemical inducesgene expression, or a chemical-repressible promoter, where applicationof the chemical represses gene expression. Chemical-inducible promotersare known in the art and include, but are not limited to, the maizeIn2-2 promoter, which is activated by benzenesulfonamide herbicidesafeners; the maize GST promoter, which is activated by hydrophobicelectrophilic compounds that are used as pre-emergent herbicides; andthe tobacco PR-1a promoter, which is activated by salicylic acid. Otherchemical-regulated promoters of interest include steroid-responsivepromoters. See, for example, the glucocorticoid-inducible promoter inSchena et al. (1991) Proc. Natl. Acad. Sci. USA 88:10421-10425 andMcNellis et al. (1998) Plant J. 14(2):247-257 and thetetracycline-inducible and tetracycline-repressible promoters forexample, Gatz et al. (1991) Mol. Gen. Genet. 227:229-237, and U.S. Pat.Nos. 5,814,618 and 5,789,156, herein incorporated by reference.

[0375] Tissue-preferred promoters can also be utilized to target GATexpression within a particular plant tissue. Tissue-preferred promotersinclude those disclosed in Yamamoto et al. (1997) Plant J.12(2):255-265; Kawamata et al. (1997) Plant Cell Physiol. 38(7):792-803;Hansen et al. (1997) Mol. Gen Genet. 254(3):337-343; Russell et al.(1997) Transgenic Res. 6(2):157-168; Rinehart et al. (1996) PlantPhysiol. 112(3):1331-1341; Van Camp et al. (1996) Plant Physiol.112(2):525-535; Canevascini et al. (1996) Plant Physiol. 112(2):513-524;Yamamoto et al. (1994) Plant Cell Physiol. 35(5):773-778; Lam (1994)Results Probl. Cell Differ. 20:181-196; Orozco et al. (1993) Plant MolBiol. 23(6):1129-1138; Matsuoka et al. (1993) Proc Natl. Acad. Sci. USA90(20):9586-9590; and Guevara-Garcia et al.(1993)Plant J. 4(3):495-505.Such promoters can be modified, if necessary, for weak expression.

[0376] Leaf-specific promoters are known in the art. See, for example,Yamamoto et al. (1997) Plant J. 12(2):255-265; Kwon et al. (1994) PlantPhysiol. 105:357-67; Yamamoto et al. (1994) Plant Cell Physiol.35(5):773-778; Gotor et al. (1993) Plant J. 3:509-18; Orozco et al.(1993) Plant Mol. Biol. 23(6):1129-1138; and Matsuoka et al. (1993)Proc. Natl. Acad. Sci. USA 90(20):9586-9590.

[0377] Root-preferred promoters are known and can be selected from themany available from the literature or isolated de novo from variouscompatible species. See, for example, Hire et al. (1992) Plant Mol.Biol. 20(2):207-218 (soybean root-specific glutamine synthetase gene);Keller et al. (1991) Plant Cell 3(10):1051-1061 (root-specific controlelement in the GRP 1.8 gene of French bean); Sanger et al. (1990) PlantMol. Biol. 14(3):433-443 (root-specific promoter of the mannopinesynthase (MAS) gene of Agrobacterium tumefaciens); and Miao et al.(1991) Plant Cell 3(1):11-22 (full-length cDNA clone encoding cytosolicglutamine synthetase (GS), which is expressed in roots and root nodulesof soybean). See also Bogusz et al. (1990) Plant Cell 2(7):633-641,which discloses two root-specific promoters isolated from hemoglobingenes from the nitrogen-fixing nonlegume Parasponia andersonii and therelated non-nitrogen-fixing nonlegume Trema tomentosa. The promoters ofthese genes were linked to a β-glucuronidase reporter gene andintroduced into both the nonlegume Nicotiana tabacum and the legumeLotus corniculatus, and in both instances root-specific promoteractivity was preserved. Leach et al. (1991) describe their analysis ofthe promoters of the highly expressed rolC and rolD root-inducing genesof Agrobacterium rhizogenes (see Plant Science (Limerick) 79(1):69-76).They concluded that enhancer and tissue-preferred DNA determinants aredissociated in those promoters. Teeri et al. (1989) EMBO J. 8(2):343-350used gene fusion to lacZ to show that the Agrobacterium T-DNA geneencoding octopine synthase is especially active in the epidermis of theroot tip and that the TR2′ gene is root specific in the intact plant andstimulated by wounding in leaf tissue, which is an especially desirablecombination of characteristics for use with an insecticidal orlarvicidal gene. The TR1′ gene, fused to nptII (neomycinphosphotransferase II), showed similar characteristics. Additionalroot-preferred promoters include the VfENOD-GRP3 gene promoter (Kusteret al. (1995) Plant Mol. Biol. 29(4):759-772); the ZRP2 promoter (U.S.Pat. No. 5,633,636); the IFS1 promoter (U.S. patent application Ser. No.10/104,706) and the rolB promoter (Capana et al. (1994) Plant Mol. Biol.25(4):681-691). See also U.S. Pat. Nos. 5,837,876; 5,750,386; 5,459,252;5,401,836; 5,110,732; and 5,023,179.

[0378] “Seed-preferred” promoters include both “seed-specific” promoters(those promoters active during seed development such as promoters ofseed storage proteins) as well as “seed-germinating” promoters (thosepromoters active during seed germination). See Thompson et al. (1989)BioEssays 10:108, herein incorporated by reference. Such seed-preferredpromoters include, but are not limited to, Cim1 (cytokinin-inducedmessage); cZ19B1 (maize 19 kDa zein); milps (myo-inositol-1-phosphatesynthase); and celA (cellulose synthase) (see U.S. Pat. No. 6,225,529,herein incorporated by reference). Gamma-zein is an endosperm-specificpromoter. Glob-1 is an embryo-specific promoter. For dicots,seed-specific promoters include, but are not limited to, beanβ-phaseolin, napin, β-conglycinin, soybean lectin, cruciferin, and thelike. For mono cots, seed-specific promoters include, but are notlimited to, maize 15 kDa zein, 22 kDa zein, 27 kDa zein, g-zein, waxy,shrunken 1, shrunken 2, globulin 1, etc. See also WO 00/12733, whichdiscloses seed-preferred promoters from end1 and end2 genes; hereinincorporated by reference.

[0379] In particular, a strongly or weakly constitutive plant promoterthat directs expression of a GAT nucleic acid in all tissues of a plantcan be favorably employed. Such promoters are active under mostenvironmental conditions and states of development or celldifferentiation. In addition to the promoters mentioned above examplesof constitutive promoters include the 1′- or 2′-promoter ofAgrobacterium tumefaciens, and other transcription initiation regionsfrom various plant genes known to those of skill. Where over expressionof a GAT polypeptide of the invention is detrimental to the plant, oneof skill will recognize that weak constitutive promoters can be used forlow-levels of expression. Generally, by “weak promoter” a promoter thatdrives expression of a coding sequence at a low level is intended. By“low level” levels from about {fraction (1/1000)} transcripts to about{fraction (1/100,000)}, transcripts to as low as about {fraction(1/500,000)} transcripts per cell are intended. Alternatively, it isrecognized that weak promoters also include promoters that are expressedin only a few cells and not in others to give a total low level ofexpression. Where a promoter is expressed at unacceptably high levels,portions of the promoter sequence can be deleted or modified to decreaseexpression levels. In those cases where high levels of expression is notharmful to the plant, a strong promoter, e.g., a t-RNA, or other pol IIIpromoter, or a strong pol II promoter, (e.g., the cauliflower mosaicvirus promoter, CaMV, 35S promoter) can be used.

[0380] Alternatively, a plant promoter can be under environmentalcontrol. Such promoters are referred to as “inducible” promoters.Examples of environmental conditions that may alter transcription byinducible promoters include pathogen attack, anaerobic conditions, orthe presence of light. In some cases, it is desirable to use promotersthat are “tissue-specific” and/or are under developmental control suchthat the GAT polynucleotide is expressed only in certain tissues orstages of development, e.g., leaves, roots, shoots, etc. Endogenouspromoters of genes related to herbicide tolerance and related phenotypesare particularly useful for driving expression of GAT nucleic acids,e.g., P450 monooxygenases, glutathione-S-transferases,homoglutathione-S-transferases, glyphosate oxidases and5-enolpyruvylshikimate-2-phosphate synthases.

[0381] Tissue specific promoters can also be used to direct expressionof heterologous structural genes, including the GAT polynucleotidesdescribed herein. Thus the promoters can be used in recombinantexpression cassettes to drive expression of any gene whose expression isdesirable in the transgenic plants of the invention, e.g., GAT and/orother genes conferring herbicide resistance or tolerance, genes whichinfluence other useful characteristics, e.g., heterosis. Similarly,enhancer elements, e.g., derived from the 5′ regulatory sequences orintron of a heterologous gene, can also be used to improve expression ofa heterologous structural gene, such as a GAT polynucleotide.

[0382] In general, the particular promoter used in the expressioncassette in plants depends on the intended application. Any of a numberof promoters which direct transcription in plant cells can be suitable.The promoter can be either constitutive or inducible. In addition to thepromoters noted above, promoters of bacterial origin which operate inplants include the octopine synthase promoter, the nopaline synthasepromoter and other promoters derived from Ti plasmids. See,Herrera-Estrella et al. (1983) Nature 303:209. Viral promoters includethe 35S and 19S RNA promoters of CaMV. See, Odell et al., (1985) Nature313:810. Other plant promoters include the ribulose-1,3-bisphosphatecarboxylase small subunit promoter and the phaseolin promoter. Thepromoter sequence from the E8 gene (see, Deikman and Fischer (1988) EMBOJ 7:3315) and other genes are also favorably used. Promoters specificfor monocotyledonous species are also considered (McElroy D., BrettellR. I. S. 1994. Foreign gene expression in transgenic cereals. TrendsBiotech., 12:62-68.) Alternatively, novel promoters with usefulcharacteristics can be identified from any viral, bacterial, or plantsource by methods, including sequence analysis, enhancer or promotertrapping, and the like, known in the art.

[0383] In preparing expression vectors of the invention, sequences otherthan the promoter and the GAT encoding gene are also favorably used. Ifproper polypeptide expression is desired, a polyadenylation region canbe derived from the natural gene, from a variety of other plant genes,or from T-DNA. Signal/localization peptides, which, e.g., facilitatetranslocation of the expressed polypeptide to internal organelles (e.g.,chloroplasts) or extracellular secretion, can also be employed.

[0384] The vector comprising the GAT polynucleotide also can include amarker gene which confers a selectable phenotype on plant cells. Forexample, the marker may encode biocide tolerance, particularlyantibiotic tolerance, such as tolerance to kanamycin, G418, bleomycin,hygromycin, or herbicide tolerance, such as tolerance to chlorosulfuron,or phophinothricin. Reporter genes, which are used to monitor geneexpression and protein localization via visualizable reaction products(e.g., beta-glucuronidase, beta-galactosidase, and chloramphenicolacetyltransferase) or by direct visualization of the gene product itself(e.g., green fluorescent protein, GFP; Sheen et al. (1995) The PlantJournal 8:777) can be used for, e.g., monitoring transient geneexpression in plant cells. Transient expression systems can be employedin plant cells, for example, in screening plant cell cultures forherbicide tolerance activities.

[0385] Plant Transformation

[0386] Protoplasts

[0387] Numerous protocols for establishment of transformable protoplastsfrom a variety of plant types and subsequent transformation of thecultured protoplasts are available in the art and are incorporatedherein by reference. For examples, see, Hashimoto et al. (1990) PlantPhysiol. 93:857; Fowke and Constabel (eds.)(1994) Plant Protoplasts;Saunders et al. (1993) Applications of Plant In Vitro TechnologySymposium, UPM 16-18; and Lyznik et al. (1991) BioTechniques 10:295,each of which is incorporated herein by reference.

[0388] Chloroplasts

[0389] Chloroplasts are a site of action of some herbicide toleranceactivities, and, in some instances, the GAT polynucleotide is fused to achloroplast transit sequence peptide to facilitate translocation of thegene products into the chloroplasts. In these cases, it can beadvantageous to transform the GAT polynucleotide into the chloroplastsof the plant host cells. Numerous methods are available in the art toaccomplish chloroplast transformation and expression (e.g., Daniell etal. (1998) Nature Biotechnology 16:346; O'Neill et al. (1993) The PlantJournal 3:729; and Maliga (1993) TIBTECH 11:1). The expression constructcomprises a transcriptional regulatory sequence functional in plantsoperably linked to a polynucleotide encoding the GAT polypeptide.Expression cassettes that are designed to function in chloroplasts (suchas an expression cassette including a GAT polynucleotide) include thesequences necessary to ensure expression in chloroplasts. Typically, thecoding sequence is flanked by two regions of homology to thechloroplastid genome to effect a homologous recombination with thechloroplast genome; often a selectable marker gene is also presentwithin the flanking plastid DNA sequences to facilitate selection ofgenetically stable transformed chloroplasts in the resultanttransplastonic plant cells (see, e.g., Maliga (1993) and Daniell (1998)supra, and references cited therein).

[0390] General Transformation Methods

[0391] DNA constructs of the invention can be introduced into the genomeof the desired plant host by a variety of conventional techniques.Techniques for transforming a wide variety of higher plant species arewell known and described in the technical and scientific literature.See, e.g., Payne, Gamborg, Croy, Jones, etc. all supra, as well as,e.g., Weising et al. (1988) Ann. Rev. Genet. 22:421 and U.S. Pat. Nos.5,889,191, 5,889,190, 5,866,785, 5,589,367 and 5,316,931, hereinincorporated by reference.

[0392] A variety of other transformation protocols are contemplated inthe present invention. Transformation protocols as well as protocols forintroducing nucleotide sequences into plants may vary depending on thetype of plant or plant cell, i.e., monocot or dicot, targeted fortransformation. Suitable methods of introducing nucleotide sequencesinto plant cells and subsequent insertion into the plant genome includemicroinjection (Crossway et al. (1986) Biotechniques 4:320-334),electroporation (Riggs et al. (1986) Proc. Natl. Acad. Sci. USA83:5602-5606), Agrobacterium-mediated transformation (U.S. Pat. Nos.5,563,055 and 5,981,840), direct gene transfer (Paszkowski et al. (1984)EMBO J. 3:2717-2722), and ballistic particle acceleration (see, forexample, U.S. Pat. Nos. 4,945,050; U.S. Pat. Nos. 5,879,918; 5,886,244;5,932,782; Tomes et al. (1995) “Direct DNA Transfer into Intact PlantCells via Microprojectile Bombardment,” in Plant Cell, Tissue, and OrganCulture: Fundamental Methods, Eds., Gamborg and Phillips(Springer-Verlag, Berlin); McCabe et al. (1988) Biotechnology6:923-926); and Lec1 transformation (WO 00/28058). See also, Weissingeret al. (1988) Ann. Rev. Genet. 22:421-477; Sanford et al. (1987)Particulate Science and Technology 5:27-37 (onion); Christou et al.(1988) Plant Physiol. 87:671-674 (soybean); McCabe et al. (1988)Bio/Technology 6:923-926 (soybean); Finer and McMullen (1991) In VitroCell Dev. Biol. 27P:175-182 (soybean); Singh et al. (1998) Theor. Appl.Genet. 96:319-324 (soybean); Datta et al. (1990) Biotechnology 8:736-740(rice); Klein et al. (1988) Proc. Natl. Acad. Sci. USA 85:4305-4309(maize); Klein et al. (1988) Biotechnology 6:559-563 (maize); U.S. Pat.Nos. 5,240,855; 5,322,783 and 5,324,646; Klein et al. (1988) PlantPhysiol. 91:440-444 (maize); Fromm et al. (1990) Biotechnology 8:833-839(maize); Hooykaas-Van Slogteren et al. (1984) Nature (London)311:763-764; U.S. Pat. No. 5,736,369 (cereals); Bytebier et al. (1987)Proc. Natl. Acad. Sci. USA 84:5345-5349 (Liliaceae); De Wet et al.(1985) in The Experimental Manipulation of Ovule Tissues, Eds., Chapmanet al. (Longman, N.Y.), pp. 197-209 (pollen); Kaeppler et al. (1990)Plant Cell Reports 9:415-418 and Kaeppler et al. (1992) Theor. Appl.Genet. 84:560-566 (whisker-mediated transformation); D'Halluin et al.(1992) Plant Cell 4:1495-1505 (electroporation); Li et al. (1993) PlantCell Reports 12:250-255 and Christou and Ford (1995) Annals of Botany75:407-413 (rice); Osjoda et al. (1996) Nature Biotechnology 14:745-750(maize via Agrobacterium tumefaciens); all of which are hereinincorporated by reference.

[0393] For example, DNAs can be introduced directly into the genomic DNAof a plant cell using techniques such as electroporation andmicroinjection of plant cell protoplasts, or the DNA constructs can beintroduced directly to plant tissue using ballistic methods, such as DNAparticle bombardment. Alternatively, the DNA constructs can be combinedwith suitable T-DNA flanking regions and introduced into a conventionalAgrobacterium tumefaciens host vector. The virulence functions of theAgrobacterium host will direct the insertion of the construct andadjacent marker into the plant cell DNA when the plant cell is infectedby the bacteria.

[0394] Microinjection techniques are known in the art and well describedin the scientific and patent literature. The introduction of DNAconstructs using polyethylene glycol precipitation is described inPaszkowski et al (1984) EMBO J 3:2717. Electroporation techniques aredescribed in Fromm et al. (1985) Proc Nat'l Acad Sci USA 82:5824.Ballistic transformation techniques are described in Klein et al. (1987)Nature 327:70; and Weeks et al. Plant Physiol 102:1077.

[0395] In some embodiments, Agrobacterium mediated transformationtechniques are used to transfer the GAT sequences of the invention totransgenic plants. Agrobacterium-mediated transformation is widely usedfor the transformation of dicots, however, certain monocots can also betransformed by Agrobacterium. For example, Agrobacterium transformationof rice is described by Hiei et al. (1994) Plant J. 6:271; U.S. Pat. No.5,187,073; U.S. Pat. No. 5,591,616; Li et al. (1991) Science in China34:54; and Raineri et al. (1990) Bio/Technology 8:33. Transformed maize,barley, triticale and asparagus by Agrobacterium mediated transformationhave also been described (Xu et al. (1990) Chinese J Bot 2:81).

[0396] Agrobacterium mediated transformation techniques take advantageof the ability of the tumor-inducing (Ti) plasmid of A. tumefaciens tointegrate into a plant cell genome, to co-transfer a nucleic acid ofinterest into a plant cell. Typically, an expression vector is producedwherein the nucleic acid of interest, such as a GAT polynucleotide ofthe invention, is ligated into an autonomously replicating plasmid whichalso contains T-DNA sequences. T-DNA sequences typically flank theexpression cassette nucleic acid of interest and comprise theintegration sequences of the plasmid. In addition to the expressioncassette, T-DNA also typically includes a marker sequence, e.g.,antibiotic resistance genes. The plasmid with the T-DNA and theexpression cassette are then transfected into Agrobacterium cells.Typically, for effective transformation of plant cells, the A.tumefaciens bacterium also possesses the necessary vir regions on aplasmid, or integrated into its chromosome. For a discussion ofAgrobacterium mediated transformation, see, Firoozabady and Kuehnle,(1995) Plant Cell Tissue and Organ Culture Fundamental Methods, Gamborgand Phillips (eds.).

[0397] Regeneration of Transgenic Plants

[0398] Transformed plant cells which are derived by plant transformationtechniques, including those discussed above, can be cultured toregenerate a whole plant which possesses the transformed genotype (i.e.,a GAT polynucleotide), and thus the desired phenotype, such as acquiredresistance (i.e., tolerance) to glyphosate or a glyphosate analog. Suchregeneration techniques rely on manipulation of certain phytohormones ina tissue culture growth medium, typically relying on a biocide and/orherbicide marker which has been introduced together with the desirednucleotide sequences. For transformation and regeneration of maize see,Gordon-Kamm et al., The Plant Cell, 2:603-618 (1990). Alternatively,selection for glyphosate resistance conferred by the GAT polynucleotideof the invention can be performed. Plant regeneration from culturedprotoplasts is described in Evans et al. (1983) Protoplasts Isolationand Culture, Handbook of Plant Cell Culture, pp 124-176, MacmillanPublishing Company, New York; and Binding (1985) Regeneration of Plants,Plant Protoplasts pp 21-73, CRC Press, Boca Raton. Regeneration can alsobe obtained from plant callus, explants, organs, or parts thereof. Suchregeneration techniques are described generally in Klee et al. (1987)Ann Rev of Plant Phys 38:467. See also, e.g., Payne and Gamborg.

[0399] Transformed plant cells, calli or explant can be cultured onregeneration medium in the dark for several weeks, generally about 1 to3 weeks to allow the somatic embryos to mature. Preferred regenerationmedia include media containing MS salts. The plant cells, calli orexplant are then typically cultured on rooting medium in a light/darkcycle until shoots and roots develop. Methods for plant regeneration areknown in the art and preferred methods are provided by Kamo et al.,(Bot. Gaz. 146(3):324-334, 1985); West et al., (The Plant Cell5:1361-1369, 1993); and Duncan et al. (Planta 165:322-332, 1985).

[0400] Small plantlets can then be transferred to tubes containingrooting medium and allowed to grow and develop more roots forapproximately another week. The plants can then be transplanted to soilmixture in pots in the greenhouse.

[0401] The regeneration of plants containing the foreign gene introducedby Agrobacterium can be achieved as described by Horsch et al., Science,227:1229-1231 (1985) and Fraley et al., Proc. Natl. Acad. Sci. U.S.A.,80:4803 (1983). This procedure typically produces shoots within two tofour weeks and these transformant shoots are then transferred to anappropriate root-inducing medium containing the selective agent and anantibiotic to prevent bacterial growth. Transgenic plants of the presentinvention may be fertile or sterile.

[0402] Regeneration can also be obtained from plant callus, explants,organs, or parts thereof. Such regeneration techniques are describedgenerally in Klee et al., Ann. Rev. of Plant Phys. 38:467-486 (1987).The regeneration of plants from either single plant protoplasts orvarious explants is well known in the art. See, for example, Methods forPlant Molecular Biology, A. Weissbach and H. Weissbach, eds., AcademicPress, Inc., San Diego, Calif. (1988). For maize cell culture andregeneration see generally, The Maize Handbook, Freeling and Walbot,eds., Springer, N.Y. (1994); Corn and Corn Improvement, 3^(rd) Ed.,Sprague and Dudley eds., American Society of Agronomy, Madison, Wis.(1988).

[0403] After transformation with Agrobacterium, the explants typicallyare transferred to selection medium. One of skill will realize that theselection medium depends on the selectable marker that wasco-transfected into the explants. After a suitable length of time,transformants will begin to form shoots. After the shoots are about 1-2cm in length, the shoots should be transferred to a suitable root andshoot medium. Selection pressure should be maintained in the root andshoot medium.

[0404] Typically, the transformants will develop roots in about 1-2weeks and form plantlets. After the plantlets are about 3-5 cm inheight, they are placed in sterile soil in fiber pots. Those of skill inthe art will realize that different acclimation procedures are used toobtain transformed plants of different species. For example, afterdeveloping a root and shoot, cuttings, as well as somatic embryos oftransformed plants, are transferred to medium for establishment ofplantlets. For a description of selection and regeneration oftransformed plants, see, e.g., Dodds and Roberts (1995) Experiments inPlant Tissue Culture, 3^(rd) Ed., Cambridge University Press.

[0405] There are also methods for Agrobacterium transformation ofArabidopsis using vacuum infiltration (Bechtold N., Ellis J. andPelletier G,, 1993, In planta Agrobacterium mediated gene transfer byinfiltration of adult Arabidopsis thaliana plants. CR Acad Sci ParisLife Sci 316:1194-1199) and simple dipping of flowering plants (Desfeux,C., Clough S. J., and Bent A. F., 2000, Female reproductive tissues arethe primary target of Agrobacterium-mediated transformation by theArabidopsis floral-dip method. Plant Physiol. 123:895-904). Using thesemethods, transgenic seed are produced without the need for tissueculture.

[0406] There are plant varieties for which effectiveAgrobacterium-mediated transformation protocols have yet to bedeveloped. For example, successful tissue transformation coupled withregeneration of the transformed tissue to produce a transgenic plant hasnot been reported for some of the most commercially relevant cottoncultivars. Nevertheless, an approach that can be used with these plantsinvolves stably introducing the polynucleotide into a related plantvariety via Agrobacterium-mediated transformation, confirmingoperability, and then transferring the transgene to the desiredcommercial strain using standard sexual crossing or back-crossingtechniques. For example, in the case of cotton, Agrobacterium can beused to transform a Coker line of Gossypium hirustum (e.g., Coker lines310, 312, 5110 Deltapine 61 or Stoneville 213), and then the transgenecan be introduced into another more commercially relevant G. hirustumcultivar by back-crossing.

[0407] The transgenic plants of this invention can be characterizedeither genotypically or phenotypically to determine the presence of theGAT polynucleotide of the invention. Genotypic analysis can be performedby any of a number of well-known techniques, including PCR amplificationof genomic DNA and hybridization of genomic DNA with specific labeledprobes. Phenotypic analysis includes, e.g., survival of plants or planttissues exposed to a selected herbicide such as glyphosate.

[0408] One of skill will recognize that after the expression cassettecontaining the GAT gene is stably incorporated in transgenic plants andconfirmed to be operable, it can be introduced into other plants bysexual crossing. Any of a number of standard breeding techniques can beused, depending upon the species to be crossed.

[0409] In vegetatively propagated crops, mature transgenic plants can bepropagated by the taking of cuttings or by tissue culture techniques toproduce multiple identical plants. Selection of desirable transgenics ismade and new varieties are obtained and propagated vegetatively forcommercial use. In seed propagated crops, mature transgenic plants canbe self crossed to produce a homozygous inbred plant. The inbred plantproduces seed containing the newly introduced heterologous nucleic acid.These seeds can be grown to produce plants that would produce theselected phenotype.

[0410] Parts obtained from the regenerated plant, such as flowers,seeds, leaves, branches, fruit, and the like are included in theinvention, provided that these parts comprise cells comprising theisolated GAT nucleic acid. Progeny and variants, and mutants of theregenerated plants are also included within the scope of the invention,provided that these parts comprise the introduced nucleic acidsequences.

[0411] Transgenic plants expressing a selectable marker can be screenedfor transmission of the GAT nucleic acid, for example, by standardimmunoblot and DNA detection techniques. Transgenic lines are alsotypically evaluated on levels of expression of the heterologous nucleicacid. Expression at the RNA level can be determined initially toidentify and quantitate expression-positive plants. Standard techniquesfor RNA analysis can be employed and include PCR amplification assaysusing oligonucleotide primers designed to amplify only the heterologousRNA templates and solution hybridization assays using heterologousnucleic acid-specific probes. The RNA-positive plants can then beanalyzed for protein expression by Western immunoblot analysis using thespecifically reactive antibodies of the present invention. In addition,in situ hybridization and immunocytochemistry according to standardprotocols can be done using heterologous nucleic acid specificpolynucleotide probes and antibodies, respectively, to localize sites ofexpression within transgenic tissue. Generally, a number of transgeniclines are usually screened for the incorporated nucleic acid to identifyand select plants with the most appropriate expression profiles.

[0412] A preferred embodiment is a transgenic plant that is homozygousfor the added heterologous nucleic acid; i.e., a transgenic plant thatcontains two added nucleic acid sequences, one gene at the same locus oneach chromosome of a chromosome pair. A homozygous transgenic plant canbe obtained by sexually mating (selfing) a heterozygous transgenic plantthat contains a single added heterologous nucleic acid, germinating someof the seed produced and analyzing the resulting plants produced foraltered cell division relative to a control plant (i.e., native,non-transgenic). Back-crossing to a parental plant and out-crossing witha non-transgenic plant are also contemplated.

[0413] Essentially any plant can be transformed with the GATpolynucleotides of the invention. Suitable plants for the transformationand expression of the novel GAT polynucleotides of this inventioninclude agronomically and horticulturally important species. Suchspecies include, but are not restricted to members of the families:Graminae (including corn, rye, triticale, barley, millet, rice, wheat,oats, etc.); Leguminosae (including pea, beans, lentil, peanut, yambean, cowpeas, velvet beans, soybean, clover, alfalfa, lupine, vetch,lotus, sweet clover, wisteria, and sweetpea); Compositae (the largestfamily of vascular plants, including at least 1,000 genera, includingimportant commercial crops such as sunflower); and Rosaciae (includingraspberry, apricot, almond, peach, rose, etc.); as well as nut plants(including, walnut, pecan, hazelnut, etc.); and forest trees (includingPinus, Quercus, Pseutotsuga, Sequoia, Populus, etc.)

[0414] Additional targets for modification by the GAT polynucleotides ofthe invention, as well as those specified above, include plants from thegenera: Agrostis, Allium, Antirrhinum, Apium, Arachis, Asparagus,Atropa, Avena (e.g., oats), Bambusa, Brassica, Bromus, Browaalia,Camellia, Cannabis, Capsicum, Cicer, Chenopodium, Chichorium, Citrus,Coffea, Coix, Cucumis, Curcubita, Cynodon, Dactylis, Datura, Daucus,Digitalis, Dioscorea, Elaeis, Eleusine, Festuca, Fragaria, Geranium,Gossypium, Glycine, Helianthus, Heterocallis, Hevea, Hordeum (e.g.,barley), Hyoscyamus, Ipomoea, Lactuca, Lens, Lilium, Linum, Lolium,Lotus, Lycopersicon, Majorana, Malus, Mangifera, Manihot, Medicago,Nemesia, Nicotiana, Onobrychis, Oryza (e.g., rice), Panicum,Pelargonium, Pennisetum (e.g., millet), Petunia, Pisum, Phaseolus,Phleum, Poa, Prunus, Ranunculus, Raphanus, Ribes, Ricinus, Rubus,Saccharum, Salpiglossis, Secale (e.g., rye), Senecio, Setaria, Sinapis,Solanum, Sorghum, Stenotaphrum, Theobroma, Trifolium, Trigonella,Triticum (e.g., wheat), Vicia, Vigna, Vitis, Zea (e.g., corn), and theOlyreae, the Pharoideae and many others. As noted, plants in the familyGraminae are particularly desirable target plants for the methods of theinvention.

[0415] Common crop plants which are targets of the present inventioninclude corn, rice, triticale, rye, cotton, soybean, sorghum, wheat,oats, barley, millet, sunflower, canola, peas, beans, lentils, peanuts,yam beans, cowpeas, velvet beans, clover, alfalfa, lupine, vetch, lotus,sweet clover, wisteria, sweetpea and nut plants (e.g., walnut, pecan,etc).

[0416] In one aspect, the invention provides a method for producing acrop by growing a crop plant that is glyphosate-tolerant as a result ofbeing transformed with a gene encoding a glyphosate N-acetyltransferase,under conditions such that the crop plant produces a crop, andharvesting the crop. Preferably, glyphosate is applied to the plant, orin the vicinity of the plant, at a concentration effective to controlweeds without preventing the transgenic crop plant from growing andproducing the crop. The application of glyphosate can be beforeplanting, or at any time after planting up to and including the time ofharvest. Glyphosate can be applied once or multiple times. The timing ofglyphosate application, amount applied, mode of application, and otherparameters will vary based upon the specific nature of the crop plantand the growing environment, and can be readily determined by one ofskill in the art. The invention further provides a crop produced by thismethod.

[0417] The invention provides for the propagation of a plant containinga GAT polynucleotide transgene. The plant can be, for example, a monocotor a dicot. In one aspect, propagation entails crossing a plantcontaining a GAT polynucleotide transgene with a second plant, such thatat least some progeny of the cross display glyphosate tolerance.

[0418] In one aspect, the invention provides a method for selectivelycontrolling weeds in a field where a crop is being grown. The methodinvolves planting crop seeds or plants that are glyphosate-tolerant as aresult of being transformed with a gene encoding a GAT, e.g., a GATpolynucleotide, and applying to the crop and any weeds a sufficientamount of glyphosate to control the weeds without a significant adverseimpact on the crop. It is important to note that it is not necessary forthe crop to be totally insensitive to the herbicide, so long as thebenefit derived from the inhibition of weeds outweighs any negativeimpact of the glyphosate or glyphosate analog on the crop or crop plant.

[0419] In another aspect, the invention provides for use of a GATpolynucleotide as a selectable marker gene. In this embodiment of theinvention, the presence of the GAT polynucleotide in a cell or organismconfers upon the cell or organism the detectable phenotypic trait ofglyphosate resistance, thereby allowing one to select for cells ororganisms that have been transformed with a gene of interest linked tothe GAT polynucleotide. Thus, for example, the GAT polynucleotide can beintroduced into a nucleic acid construct, e.g., a vector, therebyallowing for the identification of a host (e.g., a cell or transgenicplant) containing the nucleic acid construct by growing the host in thepresence of glyphosate and selecting for the ability to survive and/orgrow at a rate that is discernibly greater than a host lacking thenucleic acid construct would survive or grow. A GAT polynucleotide canbe used as a selectable marker in a wide variety of hosts that aresensitive to glyphosate, including plants, most bacteria (including E.coli), actinomycetes, yeasts, algae and fungi. One benefit of usingherbicide resistance as a marker in plants, as opposed to conventionalantibiotic resistance, is that it obviates the concern of some membersof the public that antibiotic resistance might escape into theenvironment. Some experimental data from experiments demonstrating theuse of a GAT polynucleotide as a selectable marker in diverse hostsystems are described in the Examples section of this specification.

[0420] Selection of GAT polynucleotides conferring enhanced gyphosateresistance in transgenic plants.

[0421] Libraries of GAT encoding nucleic acids diversified according tothe methods described herein can be selected for the ability to conferresistance to glyphosate in transgenic plants. Following one or morecycles of diversification and selection, the modified GAT genes can beused as a selection marker to facilitate the production and evaluationof transgenic plants and as a means of conferring herbicide resistancein experimental or agricultural plants. For example, afterdiversification of any one or more of, e.g., SEQ ID NO:1 to SEQ ID NO:5to produce a library of diversified GAT polynucleotides, an initialfunctional evaluation can be performed by expressing the library of GATencoding sequences in E. coli. The expressed GAT polypeptides can bepurified, or partially purified as described above, and screened forimproved kinetics by mass spectrometry. Following one or morepreliminary rounds of diversification and selection, the polynucleotidesencoding improved GAT polypeptides are cloned into a plant expressionvector, operably linked to, e.g., a strong constitutive promoter, suchas the CaMV 35S promoter. The expression vectors comprising the modifiedGAT nucleic acids are transformed, typically by Agrobacterium mediatedtransformation, into Arabidopsis thaliana host plants. For example,Arabidopsis hosts are readily transformed by dipping inflorescences intosolutions of Agrobacterium and allowing them to grow and set seed.Thousands of seeds are recovered in approximately 6 weeks. The seeds arethen collected in bulk from the dipped plants and germinated in soil. Inthis manner it is possible to generate several thousand independentlytransformed plants for evaluation, constituting a high throughput (HTP)plant transformation format. Bulk grown seedlings are sprayed withglyphosate and surviving seedlings exhibiting glyphosate resistancesurvive the selection process, whereas non-transgenic plants and plantsincorporating less favorably modified GAT nucleic acids are damaged orkilled by the herbicide treatment. Optionally, the GAT encoding nucleicacids conferring improved resistance to glyphosate are recovered, e.g.,by PCR amplification using T-DNA primers flanking the library inserts,and used in further diversification procedures or to produce additionaltransgenic plants of the same or different species. If desired,additional rounds of diversification and selection can be performedusing increasing concentrations of glyphosate in each subsequentselection. In this manner, GAT polynucleotides and polypeptidesconferring resistance to concentrations of glyphosate useful in fieldconditions can be obtained.

[0422] Herbicide Resistance

[0423] The present invention provides a composition comprising two ormore polynucleotides of the invention. Preferably, the GATpolynucleotides encode GAT polypeptides having different kineticparameters, i.e., a GAT variant having a lower K_(m) can be combinedwith one having a higher k_(cat). In a further embodiment, the differentGAT polynucleotides may be coupled to a chloroplast transit sequence orother signal sequence thereby providing GAT polypeptide expression indifferent cellular compartments, organdies or secretion of one or moreof the GAT polypeptides.

[0424] The mechanism of glyphosate resistance of the present inventioncan be combined with other modes of glyphosate resistance known in theart to produce plants and plant explants with superior glyphosateresistance. For example, glyphosate-tolerant plants can be produced byinserting into the genome of the plant the capacity to produce a higherlevel of 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) as morefully described in U.S. Pat. Nos. 6,248,876 B1; 5,627,061; 5,804,425;5,633,435; 5,145,783; 4,971,908; 5,312,910; 5,188,642; 4,940,835;5,866,775; 6,225,114 B1; 6,130,366; 5,310,667; 4,535,060; 4,769,061;5,633,448; 5,510,471; Re. 36,449; RE 37,287 E; and 5,491,288; andinternational publications WO 97/04103; WO 00/66746; WO 01/66704; and WO00/66747, which are incorporated herein by reference in their entiretiesfor all purposes. Glyphosate resistance is also imparted to plants thatexpress a gene that encodes a glyphosate oxido-reductase enzyme asdescribed more fully in U.S. Pat. Nos. 5,776,760 and 5,463,175, whichare incorporated herein by reference in their entireties for allpurposes.

[0425] Further, the mechanism of glyphosate resistance of the presentinvention may be combined with other modes of herbicide resistance toprovide plants and plant explants that are resistant to glyphosate andone or more other herbicides. For example, thehydroxyphenylpyruvatedioxygenases are enzymes that catalyze the reactionin which para-hydroxyphenylpyruvate (HPP) is transformed intohomogentisate. Molecules which inhibit this enzyme, and which bind tothe enzyme in order to inhibit transformation of the HPP intohomogentisate are useful as herbicides. Plants more resistant to certainherbicides are described in U.S. Pat. Nos. 6,245,968 B1; 6,268,549; and6,069,115; and international publication WO 99/23886, which areincorporated herein by reference in their entireties for all purposes.

[0426] Sulfonylurea and imidazolinone herbicides also inhibit growth ofhigher plants by blocking acetolactate synthase (ALS) or acetohydroxyacid synthase (AHAS). The production of sulfonylurea and imidazolinonetolerant plants is described more fully in U.S. Pat. Nos. 5,605,011;5,013,659; 5,141,870; 5,767,361; 5,731,180; 5,304,732; 4,761,373;5,331,107; 5,928,937; and 5,378,824; and international publication WO96/33270, which are incorporated herein by reference in their entiretiesfor all purposes.

[0427] Glutamine synthetase (GS) appears to be an essential enzymenecessary for the development and life of most plant cells. Inhibitorsof GS are toxic to plant cells. Glufosinate herbicides have beendeveloped based on the toxic effect due to the inhibition of GS inplants. These herbicides are non-selective. They inhibit growth of allthe different species of plants present, causing their totaldestruction. The development of plants containing an exogenousphosphinothricin acetyl transferase is described in U.S. Pat. Nos.5,969,213; 5,489,520; 5,550,318; 5,874,265; 5,919,675; 5,561,236;5,648,477; 5,646,024; 6,177,616 B1; and 5,879,903, which areincorporated herein by reference in their entireties for all purposes.

[0428] Protoporphyrinogen oxidase (protox) is necessary for theproduction of chlorophyll, which is necessary for all plant survival.The protox enzyme serves as the target for a variety of herbicidalcompounds. These herbicides also inhibit growth of all the differentspecies of plants present, causing their total destruction. Thedevelopment of plants containing altered protox activity which areresistant to these herbicides are described in U.S. Pat. Nos. 6,288,306B1; 6,282,837 B1; and 5,767,373; and international publication WO01/12825, which are incorporated herein by reference in their entiretiesfor all purposes.

[0429] Accordingly, the invention provides methods for selectivelycontrolling weeds in a field containing a crop that involve planting thefield with crop seeds or plants which are glyphosate-tolerant as aresult of being transformed with a gene encoding a glyphosateN-acetyltransferase, and applying to the crop and weeds in the field asufficient amount of glyphosate to control the weeds withoutsignificantly affecting the crop.

[0430] The invention further provides methods for controlling weeds in afield and preventing the emergence of glyphosate resistant weeds in afield containing a crop which involve planting the field with crop seedsor plants that are glyphosate tolerant as a result of being transformedwith a gene encoding a glyphosate-N-acetyltransferase and a geneencoding a polypeptide imparting glyphosate tolerance by anothermechanism, such as, a glyphosate-tolerant5-enolpyruvylshikimate-3-phosphate synthase and/or a glyphosate-tolerantglyphosate oxido-reductase and applying to the crop and the weeds in thefield a sufficient amount of glyphosate to control the weeds withoutsignificantly affecting the crop.

[0431] In a further embodiment the invention provides methods forcontrolling weeds in a field and preventing the emergence of herbicideresistant weeds in a field containing a crop which involve planting thefield with crop seeds or plants that are glyphosate tolerant as a resultof being transformed with a gene encoding aglyphosate-N-acetyltransferase, a gene encoding a polypeptide impartingglyphosate tolerance by another mechanism, such as, aglyphosate-tolerant 5-enolpyruvylshikimate-3-phosphate synthase and/or aglyphosate-tolerant glyphosate oxido-reductase and a gene encoding apolypeptide imparting tolerance to an additional herbicide, such as, amutated hydroxyphenylpyruvatedioxygenase, a sulfonamide-tolerantacetolactate synthase, a sulfonamide-tolerant acetohydroxy acidsynthase, an imidazolinone-tolerant acetolactate synthase, animidazolinone-tolerant acetohydroxy acid synthase, a phosphinothricinacetyl transferase and a mutated protoporphyrinogen oxidase and applyingto the crop and the weeds in the field a sufficient amount of glyphosateand an additional herbicide, such as, a hydroxyphenylpyruvatedioxygenaseinhibitor, sulfonamide, imidazolinone, bialaphos, phosphinothricin,azafenidin, butafenacil, sulfosate, glufosinate, and a protox inhibitorto control the weeds without significantly affecting the crop.

[0432] The invention further provides methods for controlling weeds in afield and preventing the emergence of herbicide resistant weeds in afield containing a crop which involve planting the field with crop seedsor plants that are glyphosate tolerant as a result of being transformedwith a gene encoding a glyphosate-N-acetyltransferase and a geneencoding a polypeptide imparting tolerance to an additional herbicide,such as, a mutated hydroxyphenylpyruvatedioxygenase, asulfonamide-tolerant acetolactate synthase, a sulfonamide-tolerantacetohydroxy acid synthase, an imidazolinone-tolerant acetolactatesynthase, an imidazolinone-tolerant acetohydroxy acid synthase, aphosphinothricin acetyl transferase and a mutated protoporphyrinogenoxidase and applying to the crop and the weeds in the field a sufficientamount of glyphosate and an additional herbicide, such as, ahydroxyphenylpyruvatedioxygenase inhibitor, sulfonamide, imidazolinone,bialaphos, phosphinothricin, azafenidin, butafenacil, sulfosate,glufosinate, and a protox inhibitor to control the weeds withoutsignificantly affecting the crop.

EXAMPLES

[0433] The following examples are illustrative and not limiting. One ofskill will recognize a variety of non-critical parameters that can bealtered to achieve essentially similar results.

Example 1 Isolating Novel Native Gat Polynucleotides

[0434] Five native GAT polynucleotides (i.e., GAT polynucleotides thatoccur naturally in a non-genetically modified organism) were discoveredby expression cloning of sequences from Bacillus strains exhibiting GATactivity. Their nucleotide sequences were determined and are providedherein as SEQ ID NO:1 to SEQ ID NO:5. Briefly, a collection ofapproximately 500 Bacillus and Pseudomonas strains were screened fornative ability to N-acetylate glyphosate. Strains were grown in LBovernight, harvested by centrifugation, permeabilized in dilute toluene,and then washed and resuspended in a reaction mix containing buffer, 5mM glyphosate, and 200 μM acetyl-CoA. The cells were incubated in thereaction mix for between 1 and 48 hours, at which time an equal volumeof methanol was added to the reaction. The cells were then pelleted bycentrifugation and the supernatant was filtered before analysis byparent ion mode mass spectrometry. The product of the reaction waspositively identified as N-acetylglyphosate by comparing the massspectrometry profile of the reaction mix to an N-acetylglyphosatestandard as shown in FIG. 2. Product detection was dependent oninclusion of both substrates (acetyl CoA and glyphosate) and wasabolished by heat denaturing the bacterial cells.

[0435] Individual GAT polynucleotides were then cloned from theidentified strains by functional screening. Genomic DNA was prepared andpartially digested with Sau3A1 enzyme. Fragments of approximately 4 Kbwere cloned into an E. coli expression vector and transformed intoelectrocompetent E. coli. Individual clones exhibiting GAT activity wereidentified by mass spectrometry following a reaction as describedpreviously except that the toluene wash was replaced by permeabilizationwith PMBS. Genomic fragments were sequenced and the putative GATpolypeptide-encoding open reading frame identified. Identity of the GATgene was confirmed by expression of the open reading frame in E. coliand detection of high levels of N-acetylglyphosate produced fromreaction mixtures.

Example 2 Characterization of a Gat Polypeptide Isolated fromB.Licheniformis Strain B6.

[0436] Genomic DNA from B. licheniformis strain B6 was purified,partially digested with Sau3A1 and fragments of 1-10 Kb were cloned intoan E. coli expression vector. A clone with a 2.5 kb insert conferred theglyphosate-N-acetyltransferase (GAT) activity on the E. coli host asdetermined with mass spectrometry analysis. Sequencing of the insertrevealed a single complete open reading frame of 441 base pairs.Subsequent cloning of this open reading frame confirmed that it encodedthe GAT enzyme. A plasmid, pMAXY2120, is shown in FIG. 4. The geneencoding the GAT enzyme of B6 was transformed into E. coli strain XL1Blue. A 10% innoculum of a saturated culture was added to Luria broth,and the culture was incubated at 37° C. for 1 hr. Expression of GAT wasinduced by the addition of IPTG at a concentration of 1 mM. The culturewas incubated a further 4 hrs, following which, cells were harvested bycentrifugation and the cell pellet stored at −80° C.

[0437] Lysis of the cells was effected by the addition of 1 ml of thefollowing buffer to 0.2 g of cells: 25 mM HEPES, pH 7.3, 100 mM KCl and10% methanol (HKM) plus 0.1 mM EDTA, 1 mM DTT, 1 mg/ml chicken egglysozyme, and a protease inhibitor cocktail obtained from Sigma and usedaccording to the manufacturer's recommendations. After 20 minutesincubation at room temperature (e.g., 22-25° C.), lysis was completedwith brief sonication. The lysate was centrifuged and the supernatantwas desalted by passage through Sephadex G25 equilibrated with HKM.Partial purification was obtained by affinity chromatography on CoAAgarose (Sigma). The column was equilibrated with HKM and the clarifiedextract was allowed to pass through under hydrostatic pressure.Non-binding proteins were removed by washing the column with HKM, andGAT was eluted with HKM containing 1 mM Coenzyme A. This procedureprovided 4-fold purification. At this stage, approximately 65% of theprotein staining observed on an SDS polyacrylamide gel loaded with crudelysate was due to GAT, with another 20% due to chloramphenicolacetyltransferase encoded by the vector.

[0438] Purification to homogeneity was obtained by gel filtration of thepartially purified protein through Superdex 75 (Pharmacia). The mobilephase was HKM, in which GAT activity eluted at a volume corresponding toa molecular radius of 17 kD. This material was homogeneous as judged byCoomassie staining of a 3 μg sample of GAT subjected to SDSpolyacrylamide gel electrophoresis on a 12% acrylamide gel, 1 mmthickness. Purification was achieved with a 6-fold increase in specificactivity.

[0439] The apparent K_(M) for glyphosate was determined on reactionmixtures containing saturating (200 μM) Acetyl CoA, varyingconcentrations of glyphosate, and 1 μM purified GAT in buffer containing5 mM morpholine adjusted to pH 7.7 with acetic acid and 20% ethyleneglycol. Initial reaction rates were determined by continuous monitoringof the hydrolysis of the thioester bond of Acetyl CoA at 235 nm (E=3.4OD/mM/cm). Hyperbolic saturation kinetics were observed (FIG. 5), fromwhich an apparent K_(M) of 2.9±0.2 (SD) mM was obtained.

[0440] The apparent K_(M) for Acetyl CoA was determined on reactionmixtures containing 5 mM glyphosate, varying concentrations of AcetylCoA, and 0.19 μM GAT in buffer containing 5 mM morpholine adjusted to pH7.7 with acetic acid and 50% methanol. Initial reaction rates weredetermined using mass spectrometric detection of N-acetyl glyphosate.Five μl were repeatedly injected into the instrument and reaction rateswere obtained by plotting reaction time vs. area of the integrated peak(FIG. 6). Hyperbolic saturation kinetics were observed (FIG. 7), fromwhich an apparent K_(M) of 2 μM was derived. From values for V_(max)obtained at a known concentration of enzyme, a k_(cat) of 6/min wascalculated.

Example 3 Mass Spectrometry (Ms) Screening Process

[0441] Sample (5 μl) was drawn from a 96-well microtiter plate at aspeed of one sample every 26 seconds and injected into the massspectrometer (Micromass Quattro LC, triple quadrupole mass spectrometer)without any separation. The sample was carried into the massspectrometer by a mobile phase of water/methanol (50:50) at a flow rateof 500 U1/min. Each injected sample was ionized by a negativeelectrospray ionization process (needle voltage, −3.5 KV; cone voltage,20 V; source temperature, 120° C.; desolvation temperature, 250° C.;cone gas flow, 90 L/Hr; and desolvation gas flow, 600 L/Hr). Themolecular ions (m/z 210) formed during this process were selected by thefirst quadrupole for performing collision induced dissociation (CID) inthe second quadrupole, where the pressure was set at 5×10⁴ mBar and thecollision energy was adjusted to 20 Ev. The third quadrupole was set foronly allowing one of the daughter ions (m/z 124) produced from theparent ions (m/z 210) to get into the detector for signal recording. Thefirst and third quadrupoles were set at unit resolution, while thephotomultiplier was operated at 650 V. Pure N-acetylglyphosate standardswere used for comparison and peak integration was used to estimateconcentrations. It was possible to detect less than 200 NmN-acetylglyphosate by this method.

Example 4 Detection of Native or Low Activity Gat Enzymes

[0442] Native or low activity GAT enzymes typically have a k_(cat) ofapproximately 1 min⁻¹ and a K_(M) for glyphosate of 1.5-10 Mm. K_(M) foracetyl CoA was typically less than 25 μM.

[0443] Bacterial cultures were grown in rich medium in deep 96-wellplates and 0.5 ml stationary phase cells were harvested bycentrifugation, washed with 5 mM morpholine acetate pH 8, andresuspended in 0.1 ml reaction mix containing 200 μM ammonium acetylCoA, 5 mM ammonium glyphosate, and 5 μg/ml PMBS (Sigma) in 5 mMmorpholine acetate, pH 8. The PMBS permeabilizes the cell membraneallowing the substrates and products to move from the cells to thebuffer without releasing the entire cellular contents. Reactions werecarried out at 25-37° C. for 1-48 hours. The reactions were quenchedwith an equal volume of 100% ethanol and the entire mixture was filteredon a 0.45 μm MAHV Multiscreen filter plate (Millipore). Samples wereanalyzed using a mass spectrometer as described above and compared tosynthetic N-acetylglyphosate standards.

Example 5 Detection of High Activity Gat Enzymes

[0444] High activity GAT enzymes typically have a k_(cat) up to 400min⁻¹ and a K_(M) below 0.1 mM glyphosate.

[0445] Genes coding for GAT enzymes were cloned into E. coli expressionvector pQE80 (Qiagen) and introduced into E. coli strain XL1 Blue(Stratagene). Cultures were grown in 150 ul rich medium (LB with 50ug/ml carbenicllin) in shallow U-bottom 96-well polystyrene plates tolate-log phase and diluted 1:9 with fresh medium containing 1 mM IPTG(USB). After 4-8 hours induction, cells were harvested, washed with 5mMmorpholine acetate pH 6.8 and resuspended in an equal volume of the samemorpholine buffer. Reactions were carried out with up to 10 ul of washedcells. At higher activity levels, the cells were first diluted up to1:200 and 5 ul was added to 100 ul reaction mix. To measure GATactivity, the same reaction mix as described for low activity was used.However, for detecting highly active GAT enzymes the glyphosateconcentration was reduced to 0.15- 0.5 mM, the pH was reduced to 6.8,and reactions were carried out for 1 hour at 37° C. Reaction workup andMS detection were as described herein.

Example 6 Purification of Gat Enzymes

[0446] Enzyme purification was achieved by affinity chromatography ofcell lysates on CoA-agarose and gel-filtration on Superdex-75.Quantities of purified GAT enzyme up to 10 mg were obtained as follows:A 100-ml culture of E. Coli carrying a GAT polynucleotide on a pQE80vector and grown overnight in LB containing 50 ug/ml carbenicillin wasused to inoculate 1 L of LB plus 50 ug/ml carbenicillin. After 1 hr,IPTG was added to 1 mM, and the culture was grown a further 6 hr. Cellswere harvested by centrifugation. Lysis was effected by suspending thecells in 25 mM HEPES (pH 7.2), 100 mM KCl, 10% methanol (HKM), 0.1 mMEDTA, 1 mM DTT, protease inhibitor cocktail supplied by Sigma-Aldrichand 1 mg/ml of chicken egg lysozyme. After 30 minutes at roomtemperature, the cells were briefly sonicated. Particulate material wasremoved by centrifugation, and the lysate was passed through a bed ofcoenzyme A-Agarose. The column was washed with several bed volumes ofHKM and GAT was eluted in 1.5 bed volumes of HKM containing 1 mM acetylCoA. GAT in the eluate was concentrated by its retention above aCentricon YM 50 ultrafiltration membrane. Further purification wasobtained by passing the protein through a Superdex 75 column through aseries of 0.6-ml injections. The peak of GAT activity eluted at a volumecorresponding to a molecular weight of 17 kD. This method resulted inpurification of GAT enzyme to homogeneity with >85% recovery. A similarprocedure was used to obtain 0.1 to 0.4 mg quantities of up to 96shuffled variants at a time. The volume of induced culture was reducedto 1 to 10 ml, coenzyme A-Agarose affinity chromatography was performedin 0.15-ml columns packed in an MAHV filter plate (Millipore) andSuperdex 75 chromatography was omitted.

Example 7 Standard Protocol for Determination of K_(CAT) and K_(M)

[0447] k_(cat) and K_(M) for glyphosate of purified protein weredetermined using a continuous spectrophotometric assay, in whichhydrolysis of the sulfoester bond of Acetyl CoA was monitored at 235 nm.Reactions were performed at ambient temperature (about 23° C.) in thewells of a 96-well assay plate, with the following components present ina final volume of 0.3 ml: 20 mM HEPES, pH 6.8, 10% ethylene glycol, 0.2mM acetyl CoA, and various concentrations of ammonium glyphosate. Incomparing the kinetics of two GAT enzymes, both enzymes were assayedunder the same conditions, e.g., both at 23° C. k_(cat) was calculatedfrom V_(max) and the enzyme concentration, determined by Bradford assay.K_(M) was calculated from the initial reaction rates obtained fromconcentrations of glyphosate ranging from 0.125 to 10 mM, using theLineweaver-Burke transformation of the Michaelis-Menten equation.k_(cat)/K_(M) was determined by dividing the value determined fork_(cat) by the value determined for K_(M).

[0448] Using this methodology, kinetic parameters for a number of GATpolypeptides exemplified herein were determined. For example, thek_(cat), K_(M) and k_(cat)/K_(M) for the GAT polypeptide correspondingto SEQ ID NO:445 have been determined to be 322 min⁻¹, 0.5 mM and 660mM⁻¹min⁻¹, respectively, using the assay conditions described above. Thek_(cat), K_(M) and k_(cat)/K_(M) for the GAT polypeptide correspondingto SEQ ID NO:457 have been determined to be 118 min⁻¹, 0.1 mM and 1184mM⁻¹min⁻¹, respectively, using the assay conditions described above. Thek_(cat), K_(M) and k_(cat)/K_(M) for the GAT polypeptide correspondingto SEQ ID NO:300 have been determined to be 296 min⁻¹, 0.65 mM and 456mM⁻¹min⁻¹, respectively, using the assay conditions described above. Oneof skill in the art can use these numbers to confirm that a GAT activityassay is generating kinetic parameters for a GAT suitable for comparisonwith the values given herein. For example, the conditions used tocompare the activity of GATs should yield the same kinetic constants forSEQ ID NO: 300, 445 and 457 (within normal experimental variance) asthose reported herein, when the conditions are used to compare a testGAT with the GAT polypeptides exemplified herein. Kinetic parameters fora number of GAT polypeptide variants were determined according to thismethodology and are provided in Table 3. TABLE 3 GAT polypeptidek_(cat), K_(M), and K_(cat)/K_(M) values. K_(M) K_(cat)/K_(M) SEQ ID NO.Clone ID K_(cat)(min⁻¹) (mM) (mM⁻¹ min⁻¹) SEQ ID NO: 263 13_10F6 48.61.3 37.4 SEQ ID NO: 264 13_12G6 52.1 1.2 43.4 SEQ ID NO: 265 14_2A5280.8 1.6 175.5 SEQ ID NO: 266 14_2C1 133.4 3.1 43 SEQ ID NO: 26714_2F11 136.9 1.7 80.6 SEQ ID NO: 268 CHIMERA 155.4 1.3 119.6 SEQ ID NO:269 10_12D7 77.3 1.8 43 SEQ ID NO: 270 10_15F4 37.6 1 37.6 SEQ ID NO:271 10_17D1 176.2 2.2 80.1 SEQ ID NO: 272 10_17F6 47.9 1.4 34.2 SEQ IDNO: 273 10_18G9 24 1.2 20 SEQ ID NO: 274 10_1H3 76.2 1.9 40.1 SEQ ID NO:275 10_20D10 86.2 1.6 53.9 SEQ ID NO: 276 10_23F2 101.3 0.9 112.5 SEQ IDNO: 277 10_2B8 108.4 1.1 98.5 SEQ ID NO: 278 10_2C7 135 1.4 96.4 SEQ IDNO: 279 10_3G5 87.4 2 43.7 SEQ ID NO: 280 10_4H7 112 1.7 65.9 SEQ ID NO:281 10_6D11 62.4 1.2 52 SEQ ID NO: 282 10_8C6 21.7 0.7 31 SEQ ID NO: 28311C3 2.8 3.1 0.9 SEQ ID NO: 284 11G3 15.6 1.7 8.9 SEQ ID NO: 285 11H31.2 1.4 0.9 SEQ ID NO: 286 12_1F9 80.4 3 26.8 SEQ ID NO: 287 12_2G9151.4 1.5 101 SEQ ID NO: 288 12_3F1 44.1 0.9 49 SEQ ID NO: 289 12_5C1089.6 1.5 59.7 SEQ ID NO: 290 12_6A10 54.7 1.1 49.7 SEQ ID NO: 291 12_6D149 1.2 40.8 SEQ ID NO: 292 12_6F9 89.1 1.9 46.9 SEQ ID NO: 293 12_6H690.5 1.6 56.5 SEQ ID NO: 294 12_7D6 53.9 1.4 38.5 SEQ ID NO: 295 12_7G11234.5 2 117.2 SEQ ID NO: 296 12F5 3.1 1.8 1.7 SEQ ID NO: 297 12G7 2.33.7 0.6 SEQ ID NO: 298 1_2H6 9.3 0.9 10.4 SEQ ID NO: 299 13_12G12 36.10.69 52.4 SEQ ID NO: 300 13_6D10 296.5 0.65 456.1 SEQ ID NO: 301 13_7A7117 0.5 234 SEQ ID NO: 302 13_7B12 68.9 1.7 40.5 SEQ ID NO: 303 13_7C148.1 1.5 32.1 SEQ ID NO: 304 13_8G6 33.7 0.61 55.2 SEQ ID NO: 305 13_9F659 1.3 45.3 SEQ ID NO: 306 14_10C9 127 0.9 141.1 SEQ ID NO: 307 14_10H3105.2 0.6 175.3 SEQ ID NO: 308 14_10H9 127.2 1.1 115.6 SEQ ID NO: 30914_11C2 108.7 1 108.7 SEQ ID NO: 310 14_12D8 62.1 1 62.1 SEQ ID NO: 31114_12H6 91.1 0.9 101.3 SEQ ID NO: 312 14_2B6 34.2 0.63 54.3 SEQ ID NO:313 14_2G11 69.4 1.4 49.6 SEQ ID NO: 314 14_3B2 68.7 0.85 80.9 SEQ IDNO: 315 14_4H8 198.8 2 99.4 SEQ ID NO: 316 14_6A8 43.7 0.78 56 SEQ IDNO: 317 14_6B10 134.7 1.4 96.2 SEQ ID NO: 318 14_6D4 256 1 256 SEQ IDNO: 319 14_7A11 197.2 3.7 53.3 SEQ ID NO: 320 14_7A1 155.8 1.6 97.4 SEQID NO: 321 14_7A9 245.9 3.2 76.9 SEQ ID NO: 322 14_7G1 136.7 0.66 207.1SEQ ID NO: 323 14_7H9 64.4 1.3 49.5 SEQ ID NO: 324 14_8F7 90.5 1.8 50.3SEQ ID NO: 325 15_10C2 69.9 0.8 87.3 SEQ ID NO: 326 15_10D6 67.1 1 67.1SEQ ID NO: 327 15_11F9 76.4 1 76.4 SEQ ID NO: 328 15_11H3 61.9 1 61.9SEQ ID NO: 329 15_12A8 77.1 1.6 48.2 SEQ ID NO: 330 15_12D6 148.6 0.74200.8 SEQ ID NO: 331 15_12D8 59.7 1.3 45.9 SEQ ID NO: 332 15_12D9 59.71.4 42.6 SEQ ID NO: 333 15_3F10 48.7 0.9 54.1 SEQ ID NO: 334 15_3G1171.5 1.2 59.6 SEQ ID NO: 335 15_4F11 80.3 0.9 89.2 SEQ ID NO: 336 15_4H393.3 1 93.3 SEQ ID NO: 337 15_6D3 85.9 1.4 61.3 SEQ ID NO: 338 15_6G1136.9 0.9 41 SEQ ID NO: 339 15_9F6 59.6 1.1 54.2 SEQ ID NO: 340 15F5 0.52.9 0.2 SEQ ID NO: 341 16A1 10.4 2.9 3.6 SEQ ID NO: 342 16H3 3.5 2.9 1.2SEQ ID NO: 343 17C12 3.2 1.4 2.3 SEQ ID NO: 344 18D6 9.6 1.2 8 SEQ IDNO: 345 19C6 2.2 1.1 2 SEQ ID NO: 346 19D5 2.2 1.7 1.3 SEQ ID NO: 34720A12 2.8 1.1 2.5 SEQ ID NO: 348 20F2 3.9 1.9 2 SEQ ID NO: 349 21E11 1.10.7 1.5 SEQ ID NO: 350 23H11 7.1 2.2 3.2 SEQ ID NO: 351 24C1 1.7 0.9 1.8SEQ ID NO: 352 24C6 2.7 1.3 2.1 SEQ ID NO: 353 24E7 8.9 0.9 9.8 SEQ IDNO: 354 2_8C3 24.8 1.5 16.6 SEQ ID NO: 355 2H3 16.1 0.9 17.7 SEQ ID NO:356 30G8 10.2 1.6 6.4 SEQ ID NO: 357 3B_10C4 24.8 1.6 15.5 SEQ ID NO:358 3B_10G7 19.6 1 19.6 SEQ ID NO: 359 3B_12B1 22.8 1.2 19 SEQ ID NO:360 3B_12D10 5.4 0.9 6 SEQ ID NO: 361 3B_2E5 16.4 1.3 12.6 SEQ ID NO:362 3C_10H3 33.9 1.1 30.8 SEQ ID NO: 363 3C_12H10 9.1 1.2 7.6 SEQ ID NO:364 3C_9H8 11.7 1 11.7 SEQ ID NO: 365 4A_1B11 23.2 1.6 15 SEQ ID NO: 3664A_1C2 20.4 1.2 17 SEQ ID NO: 367 4B_13E1 37.2 2 18.6 SEQ ID NO: 3684B_13G10 34.9 7.6 4.6 SEQ ID NO: 369 4B_16E1 17 1 17 SEQ ID NO: 3704B_17A1 19.1 1.1 17.4 SEQ ID NO: 371 4B_18F11 14.6 1.7 8.6 SEQ ID NO:372 4B_19C8 15.9 1.2 13.2 SEQ ID NO: 373 4B_1G4 3.7 1 3.7 SEQ ID NO: 3744B_21C6 11.8 0.8 14.8 SEQ ID NO: 375 4B_2H7 27 6.2 4.4 SEQ ID NO: 3764B_2H8 38.3 1.2 31.9 SEQ ID NO: 377 4B_6D8 22.7 1.5 15.2 SEQ ID NO: 3784B_7E8 20.5 1.2 17.1 SEQ ID NO: 379 4C_8C9 9 0.6 15.1 SEQ ID NO: 380 4H11.3 1.4 0.9 SEQ ID NO: 381 6_14D10 42.2 1.5 28.2 SEQ ID NO: 382 6_15G748.4 1.3 37.3 SEQ ID NO: 383 6_16A5 43.8 1.1 39.8 SEQ ID NO: 384 6_16F535.2 1 35.2 SEQ ID NO: 385 6_17C5 35.2 1.3 27.1 SEQ ID NO: 386 6_18C732.2 1.2 26.8 SEQ ID NO: 387 6_18D7 43 1.2 35.8 SEQ ID NO: 388 6_19A1086.8 1.9 45.7 SEQ ID NO: 389 6_19B6 23.9 0.7 34.2 SEQ ID NO: 390 6_19C323.1 1.4 16.5 SEQ ID NO: 391 6_19C8 74.8 2 37.4 SEQ ID NO: 392 6_20A740.4 1 40.4 SEQ ID NO: 393 6_20A9 45.1 1.3 34.7 SEQ ID NO: 394 6_20H519.5 0.8 24.3 SEQ ID NO: 395 6_21F4 24.3 0.7 34.7 SEQ ID NO: 396 6_22C947.4 3.2 14.8 SEQ ID NO: 397 6_22D9 43.9 1.3 33.8 SEQ ID NO: 398 6_22H917.4 1.1 15.9 SEQ ID NO: 399 6_23H3 43.9 1.1 39.9 SEQ ID NO: 400 6_23H746.2 1.2 38.5 SEQ ID NO: 401 6_2H1 26.6 0.9 29.5 SEQ ID NO: 402 6_3D641.7 1 41.7 SEQ ID NO: 403 6_3G3 51.9 1 51.9 SEQ ID NO: 404 6_3H2 57.2 157.2 SEQ ID NO: 405 6_4A10 55 1.1 50 SEQ ID NO: 406 6_4B1 27 1 27 SEQ IDNO: 407 6_5D11 15.2 1 15.2 SEQ ID NO: 408 6_5F11 40.1 1.9 21.1 SEQ IDNO: 409 6_5G9 35.8 1.4 25.6 SEQ ID NO: 410 6_6D5 55.3 1 55.3 SEQ ID NO:411 6_7D1 19.7 0.5 39.5 SEQ ID NO: 412 6_8H3 44.7 1 44.7 SEQ ID NO: 4136_9G11 78.4 1.3 60.3 SEQ ID NO: 414 6F1 10.1 1.8 5.6 SEQ ID NO: 4157_1C4 17.4 1.1 15.9 SEQ ID NO: 416 7_2A10 14.5 0.8 18.2 SEQ ID NO: 4177_2A11 46.8 1.1 42.6 SEQ ID NO: 418 7_2D7 54.9 1.1 49.9 SEQ ID NO: 4197_5C7 44.7 1 44.7 SEQ ID NO: 420 7_9C9 65 1 65 SEQ ID NO: 421 9_13F1034.7 0.7 49.6 SEQ ID NO: 422 9_13F1 31.6 1.1 28.7 SEQ ID NO: 423 9_15D527.6 1.2 23 SEQ ID NO: 424 9_15D8 107.3 1.1 97.6 SEQ ID NO: 425 9_15H368.7 1.9 36.2 SEQ ID NO: 426 9_18H2 25 1.1 22.7 SEQ ID NO: 427 9_20F1237.8 1 37.8 SEQ ID NO: 428 9_21C8 28.6 1.2 23.8 SEQ ID NO: 429 9_22B150.1 1.4 35.8 SEQ ID NO: 430 9_23A10 21 1 21 SEQ ID NO: 431 9_24F6 52.50.9 58.3 SEQ ID NO: 432 9_4H10 101.3 1.5 67.5 SEQ ID NO: 433 9_4H8 47.10.6 78.5 SEQ ID NO: 434 9_8H1 74.8 1.7 44 SEQ ID NO: 435 9_9H7 28 0.7 40SEQ ID NO: 436 9C6 13 2.5 5.1 SEQ ID NO: 437 9H11 4 2.3 1.7 SEQ ID NO:438 0_4B10 190 0.68 279 SEQ ID NO: 439 0_5B11 219 0.54 406 SEQ ID NO:440 0_5B3 143 0.39 367 SEQ ID NO: 441 0_5B4 180 0.6 301 SEQ ID NO: 4420_5B8 143 0.27 522 SEQ ID NO: 443 0_5C4 205 0.67 306 SEQ ID NO: 4440_5D11 224 0.67 334 SEQ ID NO: 445 0_5D3 322 0.5 660 SEQ ID NO: 4460_5D7 244 1.1 222 SEQ ID NO: 447 0_6B4 252 0.8 315 SEQ ID NO: 448 0_6D10111 0.1 1177 SEQ ID NO: 449 0_6D11 212 0.44 481 SEQ ID NO: 450 0_6F2 1750.34 516 SEQ ID NO: 451 0_6H9 228 0.47 486 SEQ ID NO: 452 10_4C10 69.60.1 695.98 SEQ ID NO: 453 10_4D5 82.72 0.1 827.16 SEQ ID NO: 454 10_4F2231.04 0.2 1155.19 SEQ ID NO: 455 10_4F9 55.39 0.1 553.93 SEQ ID NO: 45610_4G5 176.65 0.58 304.57 SEQ ID NO: 457 10_4H4 118.36 0.1 1183.6 SEQ IDNO: 458 11_3A11 55.66 0.1 556.62 SEQ ID NO: 459 11_3B1 219.97 0.63349.17 SEQ ID NO: 460 11_3B5 194.61 0.26 748.49 SEQ ID NO: 461 11_3C1249.07 0.1 490.67 SEQ ID NO: 462 11_3C3 214.02 0.22 972.81 SEQ ID NO: 46311_3C6 184.44 0.21 878.27 SEQ ID NO: 464 11_3D6 55.3 0.1 553.01 SEQ IDNO: 465 1_1G12 58.48 0.1 584.79 SEQ ID NO: 466 1_1H1 291 1.8 162 SEQ IDNO: 467 1_1H2 164 0.44 366 SEQ ID NO: 468 1_1H5 94 1.5 63 SEQ ID NO: 4691_2A12 229 1.3 176 SEQ ID NO: 470 1_2B6 138 0.58 239 SEQ ID NO: 4711_2C4 193 0.8 242 SEQ ID NO: 472 1_2D2 124 1.2 104 SEQ ID NO: 473 1_2D4182 1.2 152 SEQ ID NO: 474 1_2F8 161 1.9 85 SEQ ID NO: 475 1_2H8 1410.48 294 SEQ ID NO: 476 1_3A2 181 0.8 227 SEQ ID NO: 477 1_3D6 226 3.564 SEQ ID NO: 478 1_3F3 167 1.5 112 SEQ ID NO: 479 1_3H2 128 0.7 183 SEQID NO: 480 1_4C5 254 0.93 273 SEQ ID NO: 481 1_4D6 137 1.4 98 SEQ ID NO:482 1_4H1 236 1.2 196 SEQ ID NO: 483 1_5H5 214 0.51 419 SEQ ID NO: 4841_6F12 209 14.7 14 SEQ ID NO: 485 1_6H6 274 1.05 259 SEQ ID NO: 4863_11A10 135.41 0.17 796.55 SEQ ID NO: 487 3_14F6 188.43 0.25 753.73 SEQID NO: 488 3_15B2 104.13 0.1 1041.32 SEQ ID NO: 489 3_6A10 126.48 0.66191.64 SEQ ID NO: 490 3_6B1 263.08 0.43 611.81 SEQ ID NO: 491 3_7F9193.55 0.29 667.4 SEQ ID NO: 492 3_8G11 99.14 0.1 991.44 SEQ ID NO: 4934_1B10 77.09 0.1 770.91 SEQ ID NO: 494 5_2B3 56.75 0.1 567.5 SEQ ID NO:495 5_2D9 75.44 0.1 754.36 SEQ ID NO: 496 5_2F10 54.72 0.1 547.22 SEQ IDNO: 497 6_1A11 45.54 0.1 455.41 SEQ ID NO: 498 6_1D5 42.92 0.1 429.16SEQ ID NO: 499 6_1F11 105.76 0.1 1057.6 SEQ ID NO: 500 6_1F1 69.81 0.1698.15 SEQ ID NO: 501 6_1H10 17.01 0.1 170.11 SEQ ID NO: 502 6_1H4 85.910.1 859.12 SEQ ID NO: 503 8_1F8 82.88 0.1 828.78 SEQ ID NO: 504 8_1G267.47 0.1 674.73 SEQ ID NO: 505 8_1G3 108.9 0.1 1088.97 SEQ ID NO: 5068_1H7 101.24 0.1 1012.4 SEQ ID NO: 507 8_1H9 78.39 0.1 783.89 SEQ ID NO:508 GAT1_21F12 5.4 4.6 1.2 SEQ ID NO: 509 GAT1_24G3 4.9 3.8 1.3 SEQ IDNO: 510 GAT1_29G1 6.2 4 1.5 SEQ ID NO: 511 GAT1_32G1 4.5 3.3 1.4 SEQ IDNO: 512 GAT2_15G8 4.5 2.8 1.6 SEQ ID NO: 513 GAT2_19H8 4.1 2.8 1.5 SEQID NO: 514 GAT2_21F1 4.2 3 1.4

[0449] K_(M) for Acetyl CoA was measured using the mass spectrometrymethod with repeated sampling during the reaction. AcetylCoA andglyphosate (ammonium salts) were placed as 50-fold-concentrated stocksolutions into a well of a mass spectrometry sample plate. Reactionswere initiated with the addition of enzyme appropriately diluted in avolatile buffer such as morpholine acetate or ammonium carbonate, pH 6.8or 7.7. The sample was repeatedly injected into the instrument andinitial rates were calculated from plots of retention time and peakarea. KM was calculated as for glyphosate.

Example 8 Selection of Transformed E. Coli

[0450] An evolved GAT gene (a chimera with a native B. licheniformisribosome binding site (AACTGAAGGAGGAATCTC; SEQ ID NO:515) attacheddirectly to the 5′ end of the GAT coding sequence) was cloned into theexpression vector pQE80 (Qiagen) between the EcoRI and HindIII sites,resulting in the plasmid pMAXY2190 (FIG. 11). This eliminated the Histag domain from the plasmid and retained the B-lactamase gene conferringresistance to the antibiotics ampicillin and carbenicillin. pMAXY2190was electroporated (BioRad Gene Pulser) into XL1 Blue (Stratagene) E.coli cells. The cells were suspended in SOC rich medium and allowed torecover for one hour. The cells were then gently pelleted, washed onetime with M9 minimal media lacking aromatic amino acids (12.8 g/LNa2HPO4.7 H2O, 3.0 g/L KH2PO4, 0.5 g/L NaCl, 1.0 g/L NH4Cl, 0.4%glucose, 2 mM MgSO4, 0.1 mM CaCl2, 10 mg/L thiamine, 10 mg/L proline, 30mg/L carbenicillin), and resuspended in 20 ml of the same M9 medium.After overnight growth at 37° C. at 250 rpm, equal volumes of cells wereplated on either M9 medium or M9 plus 1 mM glyphosate medium. pQE80vector with no GAT gene was similarly introduced into E. coli cells andplated for single colonies for comparison. Table 4 presents a summary ofthe results, demonstrating that GAT activity allows selection and growthof transformed E. coli cells with less than 1% background. Note that noIPTG induction was necessary for sufficient GAT activity to allow growthof transformed cells. Transformation was verified by re-isolation ofpMAXY2190 from the E. coli cells grown in the presence of glyphosate.TABLE 4 Glyphosate selection of pMAXY2190 in E. coli Number of coloniesPlasmid M9 − glyphosate M9 + 1 mM glyphosate pMAXY2190 568 512 pQE80 3243

Example 9

[0451] Selection of Transformed Plant Cells

[0452] Agrobacterium-mediated transformation of plant cells occurs atlow efficiencies. To allow propagation of transformed cells whileinhibiting proliferation of non-transformed cells, a selectable markeris needed. Antibiotic markers for kanamycin and hygromycin and theherbicide modifying gene bar, which detoxifies the herbicidal compoundphosphinothricin, are examples of selectable markers used in plants(Methods in Molecular Biology, 1995, 49:9-18). Here we demonstrate thatGAT activity serves as an efficient selectable marker for planttransformation. An evolved GAT gene (0_(—)5B8), SEQ ID NO:190, wascloned between a plant promoter (enhanced strawberry vein banded virus)and a ubiquinone terminator and introduced into the T-DNA region of thebinary vector pMAXY3793 suitable for transformation of plant cells viaAgrobacterium tumefaciens EHA105 as shown in FIG. 12. A screenable GUSmarker was present in the T-DNA to allow confirmation of transformation.Transgenic tobacco shoots were generated using glyphosate as the onlyselecting agent.

[0453] Axillary buds of Nicotiana tabacum L. Xanthi were subcultured onhalf-strength MS medium with sucrose (1.5%) and Gelrite (0.3%) under16-h light (35-42 μEinsteins m⁻² s⁻¹, cool white fluorescent lamps) at24° C. every 2-3 weeks. Young leaves were excised from plants after 2-3weeks subculture and were cut into 3×3 mm segments. A. tumefaciensEHA105 was inoculated into LB medium and grown overnight to a density ofA600=1.0. Cells were pelleted at 4,000 rpm for 5 minutes and resuspendedin 3 volumes of liquid co-cultivation medium composed of Murashige andSkoog (MS) medium (pH 5.2) with 2 mg/L N6-benzyladenine (BA), 1% glucoseand 400 uM acetysyringone. The leaf pieces were then fully submerged in20 ml of A. tumefaciens in 100×25 mm Petri dishes for 30 min, blottedwith autoclaved filter paper, then placed on solid co-cultivation medium(0.3% Gelrite) and incubated as described above. After 3 days ofco-cultivation, 20-30 segments were transferred to basal shoot induction(BSI) medium composed of MS solid medium (pH 5.7) with 2 mg/L BA, 3%sucrose, 0.3% Gelrite, 0-200 uM glyphosate, and 400 ug/ml Timentin.

[0454] After 3 weeks, shoots were clearly evident on the explants placedon media with no glyphosate regardless of the presence or absence of theGAT gene. T-DNA transfer from both constructs was confirmed by GUShistochemical staining of leaves from regenerated shoots. Glyphosateconcentrations greater than 20 uM completely inhibited any shootformation from the explants lacking a GAT gene. Explants infected withA. tumefaciens with the GAT construct regenerated shoots at glyphosateconcentrations up to 200 uM (the highest level tested). Transformationwas confirmed by GUS histochemical staining and by PCR fragmentamplification of the GAT gene using primers annealing to the promoterand 3′ regions. The results are summarized in Table 5. Table 5 Tobaccoshoot regeneration with glyphosate selection. Glyphosate concentration %Shoot Regeneration Transferred genes 0 uM 20 uM 40 uM 80 uM 200 uM GUS100 0 0 0 0 gat and GUS 100 60 30 5 3

Example 10 Glyphosate Selection of Transformed Yeast Cells

[0455] Selection markers for yeast transformation are usuallyauxotrophic genes that allow growth of transformed cells on a mediumlacking the specific amino acid or nucleotide. Because Saccharomycescerevisiae is sensitive to glyphosate, GAT can also be used as aselectable marker. To demonstrate this, an evolved GAT gene (0_(—)6D10),SEQ ID NO:196, is cloned from the T-DNA vector pMAXY3793 (as shown inExample 9) as a PstI-ClaI fragment containing the entire coding regionand ligated into PstI-ClaI digested p424TEF (Gene, 1995, 156:119-122) asshown in FIG. 13. This plasmid contains an E. coli origin of replicationand a gene conferring carbenicillin resistance as well as a TRP 1,tryptophan auxotroph selectable marker for yeast transformation.

[0456] The GAT containing construct is transformed into E. coli XL1 Blue(Statagene) and plated on LB carbenicillin (50 ug/ml) agar medium.Plasmid DNA is prepared and used to transform yeast strain YPH499(Stratagene) using a transformation kit (Bio101). Equal amounts oftransformed cells are plated on CSM-YNB-glucose medium (Bio101) lackingall aromatic amino acids (tryptophan, tyrosine, and phenylalanine) withadded glyphosate. For comparison, p424TEF lacking the GAT gene is alsointroduced into YPH499 and plated as described. The results demonstratethat GAT activity function will as an efficient selectable marker. Thepresence of the GAT containing vector in glyphosate selected coloniescan be confirmed by re-isolation of the plasmid and restriction digestanalysis.

Example 11 Herbicide Spray Tests of Gat Expressing Tobacco Plants

[0457] Tobacco shoots generated as described in EXAMPLE 9 were excisedfrom the explants and transferred to basal root induction (BRI) mediumcomposed of half-strength Murashige and Skoog (MS) medium, pH 5.7, with1.5% sucrose, 0.3% Gelrite, 0-200 uM glyphosate and 400 ug/ml Timentin.Rooted plants and axillary shoots were clonally propagated by cuttingthe stem and transferring it to fresh BRI medium until the desirednumber of clones was obtained. Rooted plants were carefully removed fromthe solid medium. Prior to placing the plants into small pots of soil,the roots were washed to remove any remaining Gelrite. A protectiveplastic cover was kept over the plants for at least one week until theplants were well established.

[0458] To determine if GAT expressing tobacco plants could toleratesimulated field rate sprays of glyphosate, clonal lines of severalevents per GAT variant were tested. A typical test was set up asfollows: One clone from each event was sprayed with 1 ml of solutioncontaining the isopropylamine salt of glyphosate (Sigma P5671) and0.125% Triton X-100, pH 6.8 such that the amount of active ingredientsprayed was equivalent to that present in commercial glyphosateproducts. For example, to achieve 32 oz/acre (1X) of herbicidecontaining 40% active ingredient (“ai”), 2.4 ul of 40% ai formulationwas diluted into 1 ml water and sprayed on a plant in a 4-inch squarepot (16 in²). A mock application (0×) with surfactant only was alsoincluded. In some cases a second spray was applied 1-4 weeks later.Plants were kept in controlled growth rooms at 25° C. and 70% humiditywith 16 hr light.

[0459] In this example, 10 events confirmed positive for GATO-6D 10 (SEQID NO:196), ten for GATO_(—)5D3 (SEQ ID NO:193), 8 events forGATO_(—)5B8 (SEQ ID NO:190), and plants transformed with the vector only(no GAT) were clonally propagated, transferred to soil and sprayed whenplants had an average of 5 leaves. Seed-grown wild type plants were alsosprayed. After two weeks, the vector only and seed grown plants sprayedwith 0.5, 2 or 4× glyphosate stopped growing, wilted, and turned brown.Each of the transgenic GAT plants survived the spraying procedurewithout signs of glyphosate damage such as chlorosis, leaf elongation,stunting, or browning. All 0×plants were healthy, including the non-GATcontrol plants. Three weeks later all of the surviving plants weresprayed with an 8× dose. The 0× control plants died within two weeks.Again, all GAT plants survived.

[0460] Tobacco plants transformed with GAT and selected on glyphosatewere fertile. Flowering and seed set were not detectably different fromwild type plants.

Example 12 Mendelian Inheritance of Gat Gene and Glyphosate TolerantPhenotype

[0461] Mendelian inheritance of the GAT gene and glyphosate tolerantphenotype was demonstrated with transformed Arabidopsis. Columbia typeArabidopsis plants were grown and transformed by the dipping method(Clough, S J and Bent, A F, (1998) Plant J. 16(6):735-43) with aconstruct containing the GAT variant called chimera (SEQ ID NO:16). Bulkseed was collected and GAT plants were confirmed by PCR with primersspecific to the insert within the T-DNA. T1 seed from individual eventswere sown on soil with 10-30 seeds per 2-inch square pot. When the firstset of true leaves was emerging, pots were sprayed with glyphosateequivalent to 0.5 and 1× commercial product (as calculated in EXAMPLE11). After two weeks, segregation of the transgene and tolerantphenotype was evident as shown in Table 6. TABLE 6 Summary ofsegregation data for 0.5 and 1X glyphosate tolerant T1 ArabidopsisChimera event (SEQ ID NO: 16) #Survivors #Dead Segregation ratio  1 8 11  1:1.4  3 6 22   1:3.7  5 26 2 13:1  13 10 9 1:1 65 46 19 2.4:1  Vector only 0 22 — Wild-type 0 29 —

[0462] Ratios near 3:1 indicate a single segregating dominant event.Ratios greater than 3:1 indicate several segregating inserts. Ratiosless than 3:1 can be due to small sample size effects, incompletedominance, or position effects that render expression too low to conferherbicide tolerance. Compared to the controls, it was clear that the GATgene was transmitted to the T1 generation and conferred glyphosatetolerance.

Example 13 Production of Glyphosate Resistant Maize Expressing GatTransgenes

[0463] Maize plants expressing GAT variant transgenes were producedusing the methods described in U.S. Pat. No. 5,981,849, which isincorporated herein by reference. Specifically, Agrobacteriumtumefaciens vectors were constructed according to methods known in theart. Each vector contained an insert having an ubiquitin promoter andintron, a GAT variant and a PinII terminator. Maize immature embryoswere excised and infected with an Agrobacterium tumefaciens vectorcontaining the GAT variant of interest. After infection, embryos weretransferred and cultured in co-cultivation medium. After co-cultivation,the infected immature embryos were transferred onto media containing 1.0mM glyphosate (Roundup ULTRA MAXT™). This selection lasted untilactively growing putative transgenic calli were identified. The putativetransgenic callus tissues were sampled for PCR and Western assay (datanot shown) to confirm the presence of the GAT gene. The putativetransgenic callus tissues were maintained on 1.0 mM glyphosate selectionmedia for further growth and selection before plant regeneration. Atregeneration, callus tissue confirmed to be transgenic were transferredonto maturation medium containing 0.1 mM glyphosate and cultured forsomatic embryo maturation. Mature embryos were then transferred ontoregeneration medium containing 0.1 mM glyphosate for shoot and rootformation. After shoots and roots emerged, individual plantlets weretransferred into tubes with rooting medium containing 0.1 mM glyphosate.Plantlets with established shoots and roots were transplanted into potsin the greenhouse for further growth, the generation of T0 spray dataand the production of T₁ seed.

[0464] In order to evaluate the level of glyphosate resistance of thetransgenic maize plants expressing the GAT variant transgenes, T0 plantswere sprayed with glyphosate (Roundup ULTRA MAX™) in the greenhouse.Plant resistance levels were evaluated by plant discoloration scores andplant height measurements. Plant discoloration and plant height wereevaluated according to the following scales:

[0465] Discoloration score at 1, 2, 3 and 4 weeks after spray withglyphosate

[0466] 9=no leaf/stem discoloration

[0467] 7=minor leaf/stem discoloration

[0468] 5=worse leaf/stem discoloration

[0469] 3=severely discolored plant or dying plant

[0470] 1=dead plant

[0471] Plant height measurements

[0472] before spraying with glyphosate

[0473] after spraying with glyphosate at 1, 2, 3 and 4 weeks

[0474] mature plants (at tasseling)

[0475] Two plants were sent to the greenhouse from each event(independent transgenic callus) listed in Table 7. Plant 1 was kept forseed production and was not sprayed with glyphosate. Plant 2 was sprayedat 4× glyphosate (1× glyphosate=26 ounces/acre) at 14 days aftertransplanting. The T0 plant discoloration scores with 4× spray at 7 and14 days after the spray are shown in Tables 7 and 8. Height data attasseling is shown in FIG. 14. An additional experiment was performed inwhich TO plants were sprayed with 6× glyphosate. The TO plantdiscoloration scores with 6× spray at 10 days after spray are shown inTable 9. TABLE 7 Resistance Scores at 7 days after treatment with 4xglyphosate # events tested % % % constructs with 4x events @ 9 events @7 events @ <7 18534 169 30% (50) 59% (101) 11% (18) (SEQ ID NO: 196)18537  72 40% (29) 54% (39)  6% (4) (SEQ ID NO: 193) 18540 111 32% (36)61% (67)  7% (8) (SEQ ID NO: 190) total 352 33% (115) 59% (207)  8% (30)

[0476] TABLE 8 Resistance Scores at 14 days after treatment with 4xglyphosate constructs # events tested with 4x % events @ 9 18534 169 29%(49) (SEQ ID NO: 196) 18537  72 50% (36) (SEQ ID NO: 193) 18540 111 29%(32) (SEQ ID NO: 190) total 352 33% (117)

[0477] TABLE 9 Resistance Scores at 10 days after treatment with 6xglyphosate constructs # events tested with 6x % events @ 9 19286 312 51%(160) (SEQ ID NO: 323) 19288 310 52% (163) (SEQ ID NO: 91) total 622 51%(323)

Example 14 Gat is also an Acyltransferase

[0478] The ability of GAT variants (B6 (SEQ ID NO:7), 0_(—)6D10 (SEQID:448) 17-15H3 (SEQ ID NO:601), and 20-8H12 (SEQ ID NO:816)) totransfer the roup from propionyl CoA to glyphosate was tested inreaction mixtures 5mM glyphosate or no glyphosate. Propionyl CoA waspresent at 1 mM. After the reactions were terminated and the presence offree propionyl CoA was by the addition of DTNB. All variants showedglyphosate-dependent hydrolysis of propionyl CoA. These results indicatethat GAT also functions as an acyltransferase.

Example 15 T1 Studies of Glyphosate Resistant Maize Expressing GatTransgenes

[0479] Maize plants expressing GAT variant transgenes 18-28D9b (SEQ IDNO:814) and 17-15H3 (SEQ ID NO:549) were produced using the methodsdescribed in Example 13. T1 plants were used for the generation ofglyphosate field—tolerance data. The T1 plants were treated in the fieldwith four different glyphosate spray treatments (0×, 4×, 8×, and 4×+4×)for each event. The plants were sprayed at V3 and V8. Plants were scored10 days after treatment for leaf discoloration and plant heightcomparisons as described in Example 13. The T1 field spray datacorrelated well with the results previously obtained in the greenhouseas reported in Example 13. T2 seeds were collected for further studies.

Example 16 Effect of Temperature Variation on Glyphosate Tolerance ofGlyphosate Resistant Maize Expressing Gat Transgenes

[0480] Maize plants expressing GAT variant transgenes 10_(—)4F2 (SEQ IDNO:202), 17-15H3 (SEQ ID NO:549), and 18-28D9b (SEQ ID NO:814) wereproduced using the methods described in Example 13. The effect oftemperature on glyphosate tolerance was evaluated in T1 plants. The T1plants were grown in cool/cold (day 14° C., night 8° C.), warm (day 28°C., night 20° C.), and hot (day 37° C., night 20° C.) conditions. T1plants were sprayed at V2 with four different glyphosate spraytreatments (0×, 4×, 6×, and 8×). Plants were scored at 5 and 14 daysafter treatment for leaf discoloration and plant height comparisons asdescribed in Example 13. Visual observations indicated that glyphosatetolerance is not adversely effected by the range of temperatures tested.

Example 17 Production of Glyphosate Resistant Soybean Expressing GatTransgenes

[0481] Soybean plants expressing GAT variant transgenes were producedusing the method of particle gun bombardment (see Klein et al. (1987)Nature 327:70-73) using a DuPont Biolistic PDS1000/He instrument. Theselection agent used during the transformation process was hygromycin.Either the hygromycin selectable marker gene remained in the transgenicevents or the hygromycin gene was excised by methods known in the art.DNA fragments were prepared with a synthetic constitutive promoter, aGAT variant and PinII terminator. The selectable marker gene, comprisingthe 35S CaMV promoter, HPT gene and NOS terminator, was cobombarded withthe GAT gene variant as described above. Bombarded soybean embryogenicsuspension tissue was cultured for one week in the absence of selectionagent. Embryogenic suspension tissue was placed in liquid selectionmedium for 6 weeks. Putative transgenic suspension tissue was sampledfor PCR analysis to determine the presence of the GAT gene. Putativetransgenic suspension culture tissue was maintained in selection mediumfor 3 weeks to obtain enough tissue for plant regeneration. Suspensiontissue was matured for 4 weeks using standard procedures; maturedsomatic embryos were desiccated for 4-7 days and then placed ongermination induction medium for 2-4 weeks. Germinated plantlets weretransferred to soil in cell pack trays for 3 weeks for acclimatization.Plantlets were potted to 10-inch pots in the greenhouse for evaluationof glyphosate resistance.

[0482] To determine the level of glyphosate resistance of transgenicsoybeans expressing the GAT variant transgenes, TO plants were sprayedwith glyphosate (Roundup ULTRA MAX™) in the greenhouse. Plant resistancelevels were evaluated by plant discoloration scores and plant heightmeasurements.

[0483] Discoloration score at 2 weeks after spray with glyphosate

[0484] 9=no leaf/stem discoloration

[0485] 7=minor leaf/stem discoloration

[0486] 5=worse leaf/stem discoloration

[0487] 3=severely discolored plant or dying plant

[0488] 1=dead plant

[0489] One to four plants were sent to the greenhouse from eachindependent transgenic event. An additional 1-2 plants per event weregrown in controlled environment growth chambers for seed production andwere not sprayed with glyphosate. The greenhouse plants were sprayed at1×, 2×or 4× glyphosate (1× glyphosate=26 ounces/acre of RoundUp ULTRAMAX™) 3-4 weeks after transfer to soil. The T0 plant discolorationscores with 2× and 4× spray rates are shown in Table 10 and Table 11,respectively.

[0490] These results show that soybeans are effectively transformed withGAT gene variants as confirmed by PCR analysis. Transgenic soybeansexpressing GAT gene variants are resistant to glyphosate at 2× and 4×spray rates. Events surviving the 4× glyphosate spray rate do show someminor leaf discoloration however within 2 weeks of the spray test,plants recover and demonstrate normal leaf morphology. TABLE 10Resistance Scores at 10 days after treatment with 2X glyphosate. #EVENTS TESTED WITH % EVENTS @ % EVENTS @ 2X 7-8 3-6 SEQ ID NO: 193 2715% (4) 11% (3) SEQ ID NO: 824 38  8% (3) 74% (23)

[0491] TABLE 11 Resistance Scores at 10 days after treatment with 4Xglyphosate. # EVENTS TESTED WITH % EVENTS @ % EVENTS @ 4X 7-8 3-6 SEQ IDNO: 824 23 8% (2) 43% (10)

[0492] While the foregoing invention has been described in some detailfor purposes of clarity and understanding, it will be clear to oneskilled in the art from a reading of this disclosure that variouschanges in form and detail can be made without departing from the truescope of the invention. For example, all the techniques, methods,compositions, apparatus and systems described above may be used invarious combinations. The invention is intended to include all methodsand reagents described herein, as well as all polynucleotides,polypeptides, cells, organisms, plants, crops, etc., that are theproducts of these novel methods and reagents.

[0493] All publications, patents, patent applications, or otherdocuments cited in this application are incorporated by reference intheir entirety for all purposes to the same extent as if each individualpublication, patent, patent application, or other document wereindividually indicated to be incorporated by reference for all purposes.

0 SEQUENCE LISTING The patent application contains a lengthy “SequenceListing” section. A copy of the “Sequence Listing” is available inelectronic form from the USPTO web site(http://seqdata.uspto.gov/sequence.html?DocID=20040082770). Anelectronic copy of the “Sequence Listing” will also be available fromthe USPTO upon request and payment of the fee set forth in 37 CFR1.19(b)(3).

What is claimed is:
 1. An isolated or recombinant polynucleotidecomprising: (a) a nucleotide sequence encoding an amino acid sequencethat can be optimally aligned with a sequence selected from the groupconsisting of SEQ ID NO:300, SEQ ID NO:445 and SEQ ID NO:457 to generatea similarity score of at least 460, using the BLOSUM62 matrix, a gapexistence penalty of 11, and a gap extension penalty of 1; or (b) acomplementary nucleotide sequence thereof.
 2. The isolated orrecombinant polynucleotide of claim 1, wherein a polypeptide encoded bysaid polynucleotide has glyphosate-N-acetyl transferase activity.
 3. Theisolated or recombinant polynucleotide of claim 1, wherein a polypeptideencoded by said polynucleotide has glyphosate-N-acyl transferaseactivity.
 4. The isolated or recombinant polynucleotide of claim 2,wherein the polypeptide catalyzes the acetylation of glyphosate with ak_(cat)/K_(m) of at least 10 mM⁻¹ min⁻¹ for glyphosate.
 5. The isolatedor recombinant polynucleotide of claim 2, wherein the polypeptidecatalyzes the acetylation of aminomethylphosphonic acid.
 6. An isolatedor recombinant polynucleotide comprising a nucleotide sequence encodinga polypeptide having glyphosate-N-acetyltransferase activity, thepolypeptide comprising an amino acid sequence comprising at least 20contiguous amino acids of an amino acid sequence selected from the groupconsisting of SEQ ID NO:300, SEQ ID NO:445 and SEQ ID NO:457.
 7. Theisolated or recombinant polynucleotide of claim 6, wherein thepolypeptide comprises an amino acid sequence comprising at least 50contiguous amino acids of an amino acid sequence selected from the groupconsisting of SEQ ID NO:300, SEQ ID NO:445 and SEQ ID NO:457.
 8. Theisolated or recombinant polynucleotide of claim 6, wherein thepolypeptide comprises an amino acid sequence comprising at least 100contiguous amino acids of an amino acid sequence selected from the groupconsisting of SEQ ID NO:300, SEQ ID NO:445 and SEQ ID NO:457.
 9. Theisolated or recombinant polynucleotide of claim 6, wherein thepolypeptide comprises an amino acid sequence comprising about 140contiguous amino acids of an amino acid sequence selected from the groupconsisting of SEQ ID NO:300, SEQ ID NO:445 and SEQ ID NO:457.
 10. Theisolated or recombinant polynucleotide of claim 6, wherein thepolypeptide comprises an amino acid sequence selected from the groupconsisting of SEQ ID NO:300, SEQ ID NO:445 and SEQ ID NO:457.
 11. Theisolated or recombinant polynucleotide of claim 6, comprising anucleotide sequence selected from the group consisting of SEQ ID NO:48,SEQ ID NO:193 and SEQ ID NO:205.
 12. The isolated or recombinantpolynucleotide of claim 1, wherein a parental codon has been replaced bya synonymous codon that is preferentially used in plants relative to theparental codon.
 13. The isolated or recombinant polynucleotide of claim1, further comprising a nucleotide sequence encoding an N-terminalchloroplast transit peptide.
 14. A non-native variant of the isolated orrecombinant polynucleotide of claim 1, wherein one or more amino acidsof the encoded polypeptide have been mutated.
 15. A nucleic acidconstruct comprising the isolated or recombinant polynucleotide ofclaim
 1. 16. The nucleic acid construct of claim 15, further comprisinga promoter operably linked to the isolated or recombinant polynucleotideof claim 1, where the promoter is heterologous with respect to saidpolynucleotide and effective to cause sufficient expression of theencoded polypeptide to enhance the glyphosate tolerance of a plant celltransformed with said nucleic acid construct.
 17. The nucleic acidconstruct of claim 15, wherein the isolated or recombinantpolynucleotide sequence of claim 1 functions as a selectable marker. 18.The nucleic acid construct of claim 15, wherein the construct is avector.
 19. The vector of claim 18 further comprising a secondpolynucleotide sequence encoding a second polypeptide that confers adetectable phenotypic trait upon a cell or organism expressing thesecond polypeptide at an effective level.
 20. The vector of claim 19,wherein the detectable phenotypic trait functions as selectable marker.21. The vector of claim 20, wherein the detectable phenotypic trait isselected from the group consisting of herbicide resistance and a visiblemarker.
 22. The vector of claim 18, wherein the vector further comprisesa T-DNA sequence.
 23. The vector of claim 18, wherein the isolated orrecombinant polynucleotide is operably linked to a regulatory sequence.24. The vector of claim 18, wherein the vector is a plant transformationvector.
 25. An isolated or recombinant polynucleotide comprising: (a) anucleotide that hybridizes under stringent conditions over substantiallythe entire length of a nucleotide sequence that encodes an amino acidsequence selected from the group consisting of SEQ ID NO:300, SEQ IDNO:445 and SEQ ID NO:457; (b) a complementary nucleotide sequencethereof; or (c) a fragment of (a) or (b) that encodes a polypeptide haveglyphosate-N-acetyltransferase activity
 26. The isolated or recombinantpolynucleotide of claim 25, comprising a nucleotide sequence thatencodes a glyphosate-N-acetyl transferase.
 27. The isolated orrecombinant polynucleotide of claim 25, comprising a nucleotide sequencethat encodes a glyphosate-N-acyl transferase.
 28. A compositioncomprising two or more isolated or recombinant polynucleotides ofclaim
 1. 29. The composition of claim 28 comprising at least tenisolated or recombinant polynucleotides of claim
 1. 30. A cellcomprising at least one isolated or recombinant polynucleotide of claim1, wherein the polynucleotide is heterologous to the cell.
 31. The cellof claim 30, wherein the isolated or recombinant polynucleotide isoperably linked to a regulatory sequence.
 32. A cell transduced by thevector of claim
 18. 33. The cell of claim 30 or 31, wherein the cell isa transgenic plant cell.
 34. The transgenic plant cell of claim 33,wherein the transgenic plant cell expresses an exogenous polypeptidewith glyphosate-N-acetyl transferase activity.
 35. The transgenic plantcell of claim 33, wherein the transgenic plant cell expresses anexogenous polypeptide with glyphosate-N-acyl transferase activity.
 36. Atransgenic plant or transgenic plant explant comprising the cell ofclaim
 34. 37. A transgenic plant or transgenic plant explant comprisingthe cell of claim
 35. 38. The transgenic plant or transgenic plantexplant of claim 36, wherein said plant or plant explant expresses apolypeptide with glyphosate-N-acetyl transferase activity.
 39. Thetransgenic plant or transgenic plant explant of claim 37, wherein saidplant or plant explant expresses a polypeptide with glyphosate-N-acyltransferase activity.
 40. The transgenic plant or transgenic plantexplant of claim 38 , wherein said plant or plant explant is a cropplant selected from the group of genera consisting of: Eleusine,Lollium, Bambusa, Brassica, Dactylis, Sorghum, Pennisetum, Zea, Oryza,Triticum, Secale, Avena, Hordeum, Saccharum, Coix, Glycine andGossypium.
 41. The transgenic plant or transgenic plant explant of claim39, wherein said plant or plant explant is a crop plant selected fromthe group of genera consisting of: Eleusine, Lollium, Bambusa, Brassica,Dactylis, Sorghum, Pennisetum, Zea, Oryza, Triticum, Secale, Avena,Hordeum, Saccharum, Coix, Glycine and Gossypium.
 42. The transgenicplant or transgenic plant explant of claim 38, wherein said plant orplant is Arabidopsis.
 43. The transgenic plant or transgenic plantexplant of claim 39, wherein said plant or plant explant is Arabidopsis.44. The transgenic plant or transgenic plant explant of claim 38 whereinsaid plant or plant explant is Gossypium.
 45. The transgenic plant ortransgenic plant explant of claim 39 wherein said plant or plant explantis Gossypium.
 46. The transgenic plant or transgenic plant explant ofclaim 38 wherein said plant or plant explant exhibits enhancedresistance to glyphosate as compared to a wild type plant of the samespecies, strain or cultivar.
 47. The transgenic plant or transgenicplant explant of claim 39 wherein said plant or plant explant exhibitsenhanced resistance to glyphosate as compared to a wild type plant ofthe same species, strain or cultivar.
 48. A seed produced by the plantof claim
 38. 49. A seed produced by the plant of claim
 39. 50. Atransgenic plant which contains a heterologous gene which encodes aglyphosate N-acetyltransferase having a k_(cat)/K_(m) of at least 10mM⁻¹ min⁻¹ for glyphosate, wherein the plant exhibits tolerance toglyphosate applied at a level effective to inhibit the growth of thesame plant lacking the heterologous gene, without significant yieldreduction due to herbicide application.
 51. The transgenic plant ofclaim 50, wherein the glyphosate N-acetyltransferase catalyzes theacetylation of aminomethylphosphonic acid.
 52. An isolated orrecombinant polypeptide comprising an amino acid sequence that can beoptimally aligned with a sequence selected from the group consisting ofSEQ ID NO:300, SEQ ID NO:445 and SEQ ID NO:457 to generate a similarityscore of at least 460 using the BLOSUM62 matrix, a gap existence penaltyof 11, and a gap extension penalty of 1, wherein the polypeptide hasglyphosate-N-acetyl transferase activity.
 53. The isolated orrecombinant polypeptide of claim 52, wherein the polypeptide catalyzesthe acetylation of glyphosate with a k_(cat)/K_(m) of at least 10 mM⁻¹min⁻¹ for glyphosate.
 54. The isolated or recombinant polypeptide ofclaim 53, wherein the polypeptide catalyzes the acetylation ofglyphosate with a k_(cat)/K_(m) of at least 100 mM⁻¹ min⁻¹ forglyphosate.
 55. The isolated or recombinant polypeptide of claim 54,wherein the polypeptide catalyzes the acetylation ofaminomethylphosphonic acid.
 56. An isolated or recombinant polypeptidehaving glyphosate-N-acetyltransferase activity, the polypeptidecomprising an amino acid sequence comprising at least 20 contiguousamino acids of an amino acid sequence selected from the group consistingof SEQ ID NO:445 and SEQ ID NO:457.
 57. The isolated or recombinantpolypeptide of claim 56, wherein the polypeptide comprises an amino acidsequence comprising at least 50 contiguous amino acids of an amino acidsequence selected from the group consisting of SEQ ID NO:300, SEQ IDNO:445 and SEQ ID NO:457.
 58. The isolated or recombinant polypeptide ofclaim 56, wherein the polypeptide comprises an amino acid sequencecomprising at least 100 contiguous amino acids of an amino acid sequenceselected from the group consisting of SEQ ID NO:300, SEQ ID NO:445 andSEQ ID NO:457.
 59. The isolated or recombinant polypeptide of claim 56,wherein the polypeptide comprises an amino acid sequence comprisingabout 140 contiguous amino acids of an amino acid sequence selected fromthe group consisting of SEQ ID NO:300, SEQ ID NO:445 and SEQ ID NO:457.60. The isolated or recombinant polypeptide of claim 56, wherein thepolypeptide comprises an amino acid sequence selected from the groupconsisting of SEQ ID NO:300, SEQ ID NO:445 and SEQ ID NO:457.
 61. Theisolated or recombinant polynucleotide sequence of claim 52 furthercomprising an N-terminal chloroplast transit peptide.
 62. A non-nativevariant of said polypeptide of claim 52, wherein one or more amino acidsof the polypeptide have been mutated.
 63. A non-native variant of saidpolypeptide of claim 52, wherein one or more amino acids of thepolypeptide have been altered relative to a parental polypeptide. 64.The polypeptide of claim 63, wherein said polypeptide is produced by adiversity generating procedure.
 65. The polypeptide of claim 64, whereinthe diversity generating procedure comprises mutation or recombinationof at least one parental polynucleotide encoding aglyphosate-N-acetyltransferase polypeptide.
 66. The polypeptide of claim65, wherein the parental polynucleotide is a polynucleotide of claim 1.67. The polypeptide of claim 52 further comprising a secretion sequenceor a localization sequence.
 68. The polypeptide of claim 67, wherein thelocalization sequence comprises a chloroplast transit sequence.
 69. Apolypeptide which is specifically bound by a polyclonal antisera raisedagainst one or more antigen, the antigen comprising an amino acidsequence selected from the group consisting of SEQ ID NO:300, SEQ IDNO:445 and SEQ ID NO:457.
 70. A polypeptide having GAT activitycharacterized by: (a) a K_(m) for glyphosate of at least about 2 mM orless; (b) a K_(m) for acetyl CoA of at least about 200 μM or less; and(c) a k_(cat) equal to at least about 6/minute.
 71. A method ofproducing a glyphosate resistant transgenic plant or plant cellcomprising: (a) transforming a plant or plant cell with a polynucleotideencoding a glyphosate-N-acetyltransferase; and (b) optionallyregenerating a transgenic plant from the transformed plant cell.
 72. Themethod of claim 71, wherein the polynucleotide is an isolated orrecombinant polynucleotide of claim
 1. 73. The method of claim 71,wherein the polynucleotide is derived from a bacterial source.
 74. Themethod of claim 71, comprising growing the transformed plant or plantcell in a concentration of glyphosate that inhibits the growth of awild-type plant or plant cell of the same species, which concentrationdoes not inhibit the growth of the transformed plant or plant cell. 75.The method of claim 74, comprising growing the transformed plant orplant cell or progeny of the transformed plant or plant cell inincreasing concentrations of glyphosate.
 76. The method of claim 74,comprising growing the transformed plant or plant cell in aconcentration of glyphosate that is lethal to a wild-type plant or plantcell of the same species.
 77. The method of claim 72, which comprisespropagating a plant transformed with the isolated or recombinantpolynucleotide of claim
 1. 78. The method of claim 77, wherein a firstplant is propagated by crossing between the first plant and a secondplant, such that at least some progeny of the cross display glyphosatetolerance.
 79. A method for selecting a plant or plant cell containing anucleic acid construct, comprising: (a) providing a transgenic plant orplant cell containing a nucleic acid construct, wherein the nucleic acidconstruct comprises a nucleotide sequence that encodes aglyphosate-N-acetyltransferase; (b) growing the transgenic plant orplant cell in the presence of glyphosate under conditions where theglyphosate-N-acetyltransferase is expressed at an effective level,whereby the transgenic plant or plant cell grows at a rate that isdiscernibly greater than the plant or plant cell would grow if it didnot contain the nucleic acid construct.
 80. The method of claim 79,wherein the nucleic acid construct further comprises a second nucleotidesequence encoding a polypeptide and a regulatory sequence operablylinked to the second nucleotide sequence.
 81. A method for selectivelycontrolling weeds in a field containing a crop comprising: (a) plantinga field with crop seeds or plants which are glyphosate-tolerant as aresult of being transformed with a gene encoding a glyphosateN-acetyltransferase; and (b) applying to the crop and weeds in the fielda sufficient amount of glyphosate to control the weeds withoutsignificantly affecting the crop.
 82. A method of producing agenetically transformed plant that is tolerant to glyphosate,comprising: (a) inserting into the genome of a plant cell a recombinant,double-stranded DNA molecule comprising: (i) a promoter which functionsin plant cells to cause the production of an RNA sequence; (ii) astructural DNA sequence that causes the production of an RNA sequencewhich encodes a polypeptide of claim 52; and (iii) a 3′ non-translatedregion which functions in plant cells to cause the addition of a stretchof polyadenyl nucleotides to the 3′ end of the RNA sequence; where thepromoter is heterologous with respect to the structural DNA sequence andadapted to cause sufficient expression of the encoded polypeptide toenhance the glyphosate tolerance of a plant cell transformed with saidDNA molecule; b) obtaining a transformed plant cell; and c) regeneratingfrom the transformed plant cell a genetically transformed plant whichhas increased tolerance to glyphosate.
 83. A method for producing a cropcomprising: (a) growing a crop plant that is glyphosate-tolerant as aresult of being transformed with a gene encoding a glyphosateN-acetyltransferase, under conditions such that the crop plant producesa crop; and (b) harvesting a crop from the crop plant.
 84. The method ofclaim 83 further comprises applying glyphosate to the crop plant at aconcentration effective to control weeds.
 85. The method of claim 84,where the crop is cotton, corn, or soybean.
 86. The isolated orrecombinant polynucleotide of claim 1, wherein of the amino acidresidues in the amino acid sequence that correspond to the followingpositions, at least 90% conform to the following restrictions: (a) atpositions 2, 4, 15, 19, 26, 28, 31, 45, 51, 54, 86, 90, 91, 97, 103,105, 106, 114, 123, 129, 139, 144, and/or 145 the amino acid residue isBI; and (b) at positions 3,5,8, 10, 11,14, 17, 18,24,27,32,37,38,47,48,49,52,57,58,61,62,63,68,69,79,80,82,83,89,92,100,101,104,119,120,124,125,126,128,131, 143,and/or 144 the amino acid residue is B2; wherein B 1 is an amino acidselected from the group consisting of A, I, L, M, F, W, Y, and V; and B2is an amino acid selected from the group consisting of R, N, D, C, Q, E,G, H, K, P, S, andT.
 87. The isolated or recombinant polynucleotide ofclaim 1, wherein of the amino acid residues in the amino acid sequencethat correspond to the following positions, at least 80% conform to thefollowing restrictions: (a) at positions 2, 4, 15, 19, 26, 28, 51, 54,86, 90, 91, 97, 103, 105, 106, 114, 129, 139, and/or 145 the amino acidresidue is Z1; (b) at positions 31,45 and/or 64 the amino acid residueis Z2; (c) at positions 8, 36 and/or 89 the amino acid residue is Z3;(d) at positions 82, 92, 101 and/or 120 the amino acid residue is Z4;(e) at positions 3, 11, 27 and/or 79 the amino acid residue is Z5; (f)at position 123 the amino acid residue is Z1 or Z2; (g) at positions 12,33, 35, 39, 53, 59, 112, 132, 135, 140, and/or 146 the amino acidresidue is Z1 or Z3; (h) at position 30 the amino acid residue is Z1 orZ4; (i) at position 6 the amino acid residue is Z1 or Z6; (j) atpositions 81 and/or 113 the amino acid residue is Z2 or Z3; (k) atpositions 138 and/or 142 the amino acid residue is Z2 or Z4; (I) atpositions 5, 17, 24, 57, 61, 124 and/or 126 the amino acid residue isZ3, Z4, or Z6; (m) at position 104 the amino acid residue is Z3 or Z5;(o) at positions 38, 52, 62 and/or 69 the amino acid residue is Z1, Z3or Z6; (p) at positions 14, 119 and/or 144 the amino acid residue is Z1, Z2, Z4 or Z5; (q) at position 18 the amino acid residue is Z4, Z5 orZ6; (r) at positions 10, 32, 48, 63, 80 and/or 83 the amino acid residueis Z5 or Z6; (s) at position 40 the amino acid residue is Z 1, Z2 or Z3;(t) at positions 65 and/or 96 the amino acid residue is Z1, Z3, Z5 orZ6; (u) at positions 84 and/or 115 the amino acid residue is Z1, Z3 orZ4; (v) at position 93 the amino acid residue is Z2, Z3 or Z4; (w) atposition 130 the amino acid residue is Z2, Z4 or Z6; (x) at positions 47and/or 58 the amino acid residue is Z3, Z4 or Z6; (y) at positions 49,68, 100 and/or 143 the amino acid residue is Z3, Z4 or Z5; (z) atposition 131 the amino acid residue is Z3, Z5 or Z6; (aa) at positions125 and/or 128 the amino acid residue is Z4, Z5 or Z6; (ab) at position67 the amino acid residue is Z1, Z3, Z4 or Z5; (ac) at position 60 theamino acid residue is Z1, Z4, Z5 or Z6; and (ad) at position 37 theamino acid residue is Z3, Z4, Z5 or Z6; wherein Z1 is an amino acidselected from the group consisting of A, I, L, M, and V; Z2 is an aminoacid selected from the group consisting of F, W, and Y; Z3 is an aminoacid selected from the group consisting of N, Q, S, and T; Z4 is anamino acid selected from the group consisting of R, H, and K; Z5 is anamino acid selected from the group consisting of D and E; and Z6 is anamino acid selected from the group consisting of C, G, and P.
 88. Theisolated or recombinant polynucleotide of claim 1, wherein of the aminoacid residues in the amino acid sequence that correspond to thefollowing positions, at least 90% conform to the following restrictions:(a) at positions 1, 7,9, 13,20,36,42,46,50,56, 64,70,72,75,76,78,94, 98,107, 110, 117,118,121,141 and/or 144 the amino acid residue is BI; and(b) at positions 16, 21, 22, 23, 25, 29, 34, 41, 43, 44, 55, 66, 71, 73,74, 77, 85, 87, 88, 95, 99,102,108,109,111,116, 122,127,133,134,136, 137and/or 144 the amino acid residue is B2; wherein B1 is an amino acidselected from the group consisting of A, I, L, M, F, W, Y, and V; and B2is an amino acid selected from the group consisting of R, N, D, C, Q, E,G, H, K, P, S, and T.
 89. The isolated or recombinant polynucleotide ofclaim 1, wherein of the amino acid residues in the amino acid sequencethat correspond to the following positions, at least 90% conform to thefollowing restrictions: (a) at positions 1, 7,9,20,36,42,50,64,72,75,76,78,94,98, 110, 121, and/or 141 the amino acid residue is Z 1;(b) at positions 13, 46, 56, 64, 70, 107, 117, and/or 118 the amino acidresidue is Z2; (c) at positions 23, 36, 55, 71, 77, 88, and/or 109 theamino acid residue is Z3; (d) at positions 16, 21, 41, 73, 85, 99,and/or 111 the amino acid residue is Z4; (e) at positions 34 and/or 95the amino acid residue is Z5; (f) at position 22, 25, 29, 43, 44, 66,74, 87, 102, 108, 116, 122, 127, 133, 134, 136, and/or 137 the aminoacid residue is Z6; wherein Z1 is an amino acid selected from the groupconsisting of A, I, L, M, and V; Z2 is an amino acid selected from thegroup consisting of F, W, and Y; Z3 is an amino acid selected from thegroup consisting of N, Q, S, and T; Z4 is an amino acid selected fromthe group consisting of R, H, and K; Z5 is an amino acid selected fromthe group consisting of D and E; and Z6 is an amino acid selected fromthe group consisting of C, G, and P.
 90. The isolated or recombinantpolynucleotide of claim 86, wherein of the amino acid residues in theamino acid sequence that correspond to the following positions, at least90% conform to the following restrictions: (a) at positions 1, 7,9,13,20,36,42,46,50,56, 64,70, 72,75,76,78,94,98, 107, 110,117,118,121,141 and/or 144 the amino acid residue is B1; and (b) atpositions 16, 21, 22, 23, 25, 29, 34, 41, 43, 44, 55, 66, 71, 73, 74,77, 85, 87, 88, 95, 99,102,108,109,111, 116,122,127,133,134, 136,137and/or 144 the amino acid residue is B2; wherein B1 is an amino acidselected from the group consisting of A, I, L, M, F, W, Y, and V; and B2is an amino acid selected from the group consisting of R, N, D, C, Q, E,G, H, K, P, S, and T.
 91. The isolated or recombinant polynucleotide ofclaim 86, wherein of the amino acid residues in the amino acid sequencethat correspond to the following positions, at least 90% conform to thefollowing restrictions: (a) at positions 1, 7,9, 13,20,36,42,46,50,56,64,70,72,75,76,78,94, 98, 107, 110, 117, 118, 121, 141 and/or 144 theamino acid residue is B1; and (b) at positions 16, 21, 22, 23, 25, 29,34, 41, 43, 44, 55, 66, 71, 73, 74, 77, 85, 87, 88, 95, 99, 102, 108,109, 111, 116, 122, 127, 133, 134, 136, 137 and/or 144 the amino acidresidue is B2; wherein B1 is an amino acid selected from the groupconsisting of A, I, L, M, F, W, Y, and V; and B2 is an amino acidselected from the group consisting of R, N, D, C, Q, E, G, H, K, P, S,and T.
 92. The isolated or recombinant polynucleotide of claim 87,wherein of the amino acid residues in the amino acid sequence thatcorrespond to the following positions, at least 90% conform to thefollowing restrictions: (a) at positions 1, 7, 9, 13, 20, 36, 42, 46,50, 56, 64, 70, 72, 75, 76, 78, 94, 98, 107, 110, 117, 118, 121, 141and/or 144 the amino acid residue is B1 ; and (b) at positions 16, 21,22, 23, 25, 29, 34, 41, 43, 44, 55, 66, 71, 73, 74, 77, 85, 87, 88, 95,99, 102, 108, 109, 111, 116, 122, 127, 133, 134, 136, 137 and/or 144 theamino acid residue is B2; wherein B₁ is an amino acid selected from thegroup consisting of A, I, L, M, F, W, Y, and V; and B2 is an amino acidselected from the group consisting of R, N, D, C, Q, E, G,H, K, P, S,and T.
 93. The isolated or recombinant polynucleotide of claim 1,wherein of the amino acid residues in the amino acid sequence thatcorrespond to the following positions, at least 80% conform to thefollowing restrictions: (a) at position 2 the amino acid residue is I orL; (b) at position 3 the amino acid residue is E or D; (c) at position 4the amino acid residue is V, A or I; (d) at position 5 the amino acidresidue is K, R or N; (e) at position 6 the amino acid residue is P orL; (f) at position 8 the amino acid residue is N, S or T; (g) atposition 10 the amino acid residue is E or G; (h) at position 11 theamino acid residue is D or E; (i) at position 12 the amino acid residueis T or A; (j) at position 14 the amino acid residue is E, K or D; (k)at position 15 the amino acid residue is I or L; (l) at position 17 theamino acid residue is H or Q; (m) at position 18 the amino acid residueis R, C, K or E; (n) at position 19 the amino acid residue is I or V;(o) at position 24 the amino acid residue is Q or R; (p) at position 26the amino acid residue is M, V, L or I; (q) at position 27 the aminoacid residue is E or D; (r) at position 28 the amino acid residue is Aor V; (s) at position 30 the amino acid residue is K, T, M or R; (t) atposition 31 the amino acid residue is Y or F; (u) at position 32 theamino acid residue is E, G or D; (v) at position 33 the amino acidresidue is T, A or S; (w) at position 35 the amino acid residue is L, Sor M; (x) at position 37 the amino acid residue is R, G, E, Q, or C; (y)at position 38 the amino acid residue is G, S or D; (z) at position 39the amino acid residue is T, A or S; (aa) at position 40 the amino acidresidue is F, L or S; (ab) at position 45 the amino acid residue is Y orF; (ac) at position 47 the amino acid residue is R, Q or G; (ad) atposition 48 the amino acid residue is G or D; (ae) at position 49 theamino acid residue is K, R, E or Q; (af) at position 51 the amino acidresidue is I or V; (ag) at position 52 the amino acid residue is S, C orG; (ah) at position 53 the amino acid residue is I, T or V; (ai) atposition 54 the amino acid residue is A or V; (aj) at position 57 theamino acid residue is H or N; (ak) at position 58 the amino acid residueis Q, K, N, P or R; (al) at position 59 the amino acid residue is A orS; (am) at position 60 the amino acid residue is E, K, G, V or D; (an)at position 61 the amino acid residue is H or Q; (ao) at position 62 theamino acid residue is P, S, T or L; (ap) at position 63 the amino acidresidue is E, G or D; (aq) at position 65 the amino acid residue is E,D, V, Q or P; (ar) at position 67 the amino acid residue is Q, E, R, L,H or K; (as) at position 68 the amino acid residue is K, R, E, or N;(at) at position 69 the amino acid residue is Q or P; (au) at position79 the amino acid residue is E or D; (av) at position 80 the amino acidresidue is G or E; (aw) at position 81 the amino acid residue is Y, N,F,or H; (ax) at position 82 the amino acid residue is R or H; (ay) atposition 83 the amino acid residue is E, G or D; (az) at position 84 theamino acid residue is Q, R or L; (ba) at position 86 the amino acidresidue is A or V; (bb) at position 89 the amino acid residue is T, S orG; (be) at position 90 the amino acid residue is L or I; (bd) atposition 91 the amino acid residue is I, V or L; (be) at position 92 theamino acid residue is R or K; (bf) at position 93 the amino acid residueis H, Y or Q; (bg) at position 96 the amino acid residue is E, A or Q;(bh) at position 97 the amino acid residue is L or I; (bi) at position100 the amino acid residue is K, R, N or E; (bj) at position 101 theamino acid residue is K or R; (bk) at position 103 the amino acidresidue is A or V; (bl) at position 104 the amino acid residue is D orN; (bm) at position 105 the amino acid residue is L, M or I; (bn) atposition 106 the amino acid residue is L or I; (bo) at position 112 theamino acid residue is T, I or A; (bp) at position 113 the amino acidresidue is S, T or F; (bq) at position 114 the amino acid residue is Aor V; (br) at position 115 the amino acid residue is S, R or A; (bs) atposition 119 the amino acid residue is K, E or R; (bt) at position 120the amino acid residue is K or R; (bu) at position 123 the amino acidresidue is F or L; (bv) at position 124 the amino acid residue is C, Sor R; (bw) at position 125 the amino acid residue is E, K, G or D; (bx)at position 126 the amino acid residue is Q or H; (by) at position 128the amino acid residue is E, G, K or D; (bz) at position 129 the aminoacid residue is V, I or A; (ca) at position 130 the amino acid residueis Y, H, F or C; (cb) at position 131 the amino acid residue is D, G, Nor E; (cc) at position 132 the amino acid residue is I, T, A, M, V or L;(cd) at position 135 the amino acid residue is V, T, A or I; (ce) atposition 138 the amino acid residue is H or Y; (cf) at position 139 theamino acid residue is I or V; (cg) at position 140 the amino acidresidue is L, S or M; (ch) at position 142 the amino acid residue is Yor H; (ci) at position 143 the amino acid residue is K, T, E or R; (cj)at position 144 the amino acid residue is K, E, R or W; (ck) at position145 the amino acid residue is L or I; and (cl) at position 146 the aminoacid residue is T or A.
 94. The isolated or recombinant polynucleotideof claim 1, wherein of the amino acid residues in the amino acidsequence that correspond to the following positions, at least 80%conform to the following restrictions: (a) at position 9, 76, 94 and 110the amino acid residue is A; (b) at position 29 and 108 the amino acidresidue is C; (c) at position 34 the amino acid residue is D; (d) atposition 95 the amino acid residue is E; (e) at position 56 the aminoacid residue is F; (f) at position 43, 44, 66, 74, 87, 102, 116, 122,127 and 136 the amino acid residue is G; (g) at position 41 the aminoacid residue is H; (h) at position 7 the amino acid residue is I; (i) atposition 85 the amino acid residue is K; (j) at position 20, 36, 42, 50,72, 78, 98 and 121 the amino acid residue is L; (k) at position 1, 75and 141 the amino acid residue is M; (l) at position 23, 64 and 109 theamino acid residue is N; (m) at position 22, 25, 133, 134 and 137 theamino acid residue is P; (n) at position 71 the amino acid residue is Q;(o) at position 16, 21, 73, 99 and 111 the amino acid residue is R; (p)at position 55 and 88 the amino acid residue is S; (q) at position 77the amino acid residue is T; (r) at position 107 the amino acid residueis W; and (s) at position 13, 46, 70, 117 and 118 the amino acid residueis Y.
 95. The isolated or recombinant polynucleotide of claim 93,wherein of the amino acid residues in the amino acid sequence thatcorrespond to the following positions, at least 90% conform to thefollowing restrictions: (a) at positions 1, 7, 9, 13, 20, 36, 42, 46,50, 56, 64, 70, 72, 75, 76, 78, 94, 98, 107, 110, 117, 118, 121, 141and/or 144 the amino acid residue is B1; and (b) at positions 16, 21,22, 23, 25, 29, 34, 41, 43, 44, 55, 66, 71, 73, 74, 77, 85, 87, 88, 95,99, 102, 108, 109, 111, 116, 122, 127, 133, 134, 136, 137 and/or 144 theamino acid residue is B2; wherein B1 is an amino acid selected from thegroup consisting of A, I, L, M, F, W, Y, and V; and B2 is an amino acidselected from the group consisting of R, N, D, C, Q, E, G, H, K, P, S,and T.
 96. The isolated or recombinant polynucleotide of claim 94,wherein of the amino acid residues in the amino acid sequence thatcorrespond to the following positions, at least 90% conform to thefollowing restrictions: (a) at positions 2, 4, 15, 19, 26, 28, 31, 45,51, 54, 86, 90, 91, 97, 103, 105, 106, 114, 123, 129, 139, 144 and/or145 the amino acid residue is B1; and (b) at positions 3, 5, 8, 10, 11,14, 17, 18, 24, 27, 32, 37, 38, 47, 48, 49, 52, 57, 58, 61, 62,63,68,69,79,80,82,83,89,92, 100,101,104,119,120, 124,125,126,128,131,143, and/or 144 the amino acid residue is B2; wherein B1 is an aminoacid selected from the group consisting of A, I, L, M, F, W, Y, and V;and B2 is an amino acid selected from the group consisting of R, N, D,C, Q, E, G, H, K, P, S, and T.
 97. The isolated or recombinantpolynucleotide of claim 93, wherein of the amino acid residues in theamino acid sequence that correspond to the following positions, at least90% conform to the following restrictions: (a) at positions 1, 7, 9, 20,36, 42, 50, 64, 72, 75, 76, 78, 94, 98, 110, 121, and/or 141 the aminoacid residue is Z 1; (b) at positions 13, 46, 56, 64, 70, 107, 117,and/or 118 the amino acid residue is Z2; (c) at positions 23, 36, 55,71, 77, 88, and/or 109 the amino acid residue is Z3; (d) at positions16, 21, 41, 73, 85, 99, and/or 111 the amino acid residue is Z4; (e) atpositions 34 and/or 95 the amino acid residue is Z5; (f) at position 22,25, 29, 43, 44, 66, 74, 87, 102, 108, 116, 122, 127, 133, 134, 136,and/or 137 the amino acid residue is Z6; wherein Z1 is an amino acidselected from the group consisting of A, I, L, M, and V; Z2 is an aminoacid selected from the group consisting of F, W, and Y; Z3 is an aminoacid selected from the group consisting of N, Q, S, and T; Z4 is anamino acid selected from the group consisting of R, H, and K; Z5 is anamino acid selected from the group consisting of D and E; and Z6 is anamino acid selected from the group consisting of C, G, and P.
 98. Theisolated or recombinant polynucleotide of claim 94, wherein of the aminoacid residues in the amino acid sequence that correspond to thefollowing positions, at least 80% conform to the following restrictions:(a) at positions 2, 4, 15, 19, 26, 28, 51, 54, 86, 90, 91, 97, 103, 105,106, 114, 129, 139, and/or 145 the amino acid residue is Z1; (b) atpositions 31,45 and/or 64 the amino acid residue is Z2; (c) at positions8, 36 and/or 89 the amino acid residue is Z3 or Z6; (d) at positions 82,92, 101 and/or 120 the amino acid residue is Z4; (e) at positions 3, 11,27 and/or 79 the amino acid residue is Z5; (f) at position 123 the aminoacid residue is Z1 or Z2; (g) at positions 12, 33, 35, 39, 53, 59, 112,132, 135, 140, and/or 146 the amino acid residue is Z1 or Z3; (h) atposition 30 the amino acid residue is Z1 or Z4; (i) at position 6 theamino acid residue is Z1 or Z6; (j) at positions 81 and/or 113 the aminoacid residue is Z2 or Z3; (k) at positions 138 and/or 142 the amino acidresidue is Z2 or Z4; (l) at positions 5, 17, 24, 57, 61, 124 and/or 126the amino acid residue is Z3, Z4 or Z6; (m) at position 104 the aminoacid residue is Z3 or Z5; (o) at positions 38, 52, 62 and/or 69 theamino acid residue is Z1, Z3, Z5 or Z6; (p) at positions 14, 119 and/or144 the amino acid residue is Z1, Z2, Z4 or Z5; (q) at position 18 theamino acid residue is Z4, Z5 or Z6; (r) at positions 10, 32, 48, 63, 80and/or 83 the amino acid residue is Z5 or Z6; (s) at position 40 theamino acid residue is Z1, Z2 or Z3; (t) at positions 65 and/or 96 theamino acid residue is Z1, Z3, Z5 or Z6; (u) at positions 84 and/or 115the amino acid residue is Z1, Z3 or Z4; (v) at position 93 the aminoacid residue is Z2, Z3 or Z4; (w) at position 130 the amino acid residueis Z2, Z4 or Z6; (x) at positions 47 and/or 58 the amino acid residue isZ3, Z4 or Z6; (y) at positions 49, 68, 100 and/or 143 the amino acidresidue is Z3, Z4 or Z5; (z) at position 131 the amino acid residue isZ3, Z5 or Z6; (aa) at positions 125 and/or 128 the amino acid residue isZ4, Z5 or Z6; (ab) at position 67 the amino acid residue is Z1, Z3, Z4or Z5; (ac) at position 60 the amino acid residue is Z1, Z4, Z5 or Z6;and (ad) at position 37 the amino acid residue is Z3, Z4, Z5 or Z6;wherein Z₁ is an amino acid selected from the group consisting of A, I,L, M, and V; Z2 is an amino acid selected from the group consisting ofF, W, and Y; Z3 is an amino acid selected from the group consisting ofN, Q, S, and T; Z4 is an amino acid selected from the group consistingof R, H, and K; Z5 is an amino acid selected from the group consistingof D and E; and Z6 is an amino acid selected from the group consistingof C,G,andP.
 99. The isolated or recombinant polynucleotide of claim 93,wherein of the amino acid residues in the amino acid sequence thatcorrespond to the following positions, at least 80% conform to thefollowing restrictions: (a) at position 9, 76,94 and 110 the amino acidresidue is A; (b) at position 29 and 108 the amino acid residue is C;(c) at position 34 the amino acid residue is D; (d) at position 95 theamino acid residue is E; (e) at position 56 the amino acid residue is F;(f) at position 43, 44, 66, 74, 87, 102, 116, 122, 127 and 136 the aminoacid residue is G; (g) at position 41 the amino acid residue is H; (h)at position 7 the amino acid residue is I; (i) at position 85 the aminoacid residue is K; (j) at position 20, 36, 42, 50, 72, 78, 98 and 121the amino acid residue is L; (k) at position 1, 75 and 141 the aminoacid residue is M; (l) at position 23, 64 and 109 the amino acid residueis N; (m) at position 22, 25, 133, 134 and 137 the amino acid residue isP; (n) at position 71 the amino acid residue is Q; (o) at position 16,21, 73, 99 and 11 1 the amino acid residue is R; (p) at position 55 and88 the amino acid residue is S; (q) at position 77 the amino acidresidue is T; (r) at position 107 the amino acid residue is W; and (s)at position 13, 46, 70, 117 and 118 the amino acid residue is Y. 100.The isolated or recombinant polynucleotide of claim 1, wherein the aminoacid residue in the amino acid sequence that correspond to position 28is V.
 101. The isolated or recombinant polynucleotide of claim 1,wherein the amino acid sequence is selected from the group consisting ofSEQ ID NO:6-10, 263-514, 568-619, 621, 623, 625, 627, 629, 631, 633,635, 637, 639, 641, 643, 645, 647, 649, 651, 653, 655, 657, 659, 661,663, 665, 667, 669, 671, 673, 675, 677, 679, 681, 683, 685, 687,689,691,693,695,697, 699,701,703,705,707, 709,711,713,715,717,719,721,723, 725, 727, 729, 731, 733, 735, 737, 739, 741, 743, 745, 747,749, 751, 753, 755, 757, 759, 761, 763, 765, 767, 769, 771, 773, 775,777, 779, 781, 783, 785, 787, 789, 791, 793, 795, 797, 799, 801, 803,805, 807, 809, 811, and
 813. 102. The isolated or recombinantpolypeptide of claim 52, wherein of the amino acid residues in the aminoacid sequence that correspond to the following positions, at least 90%conform to the following restrictions: (a) at positions 2, 4, 15, 19,26, 28, 31, 45, 51, 54, 86, 90, 91, 97, 103, 105, 106, 114, 123, 129,139, 144 and/or 145 the amino acid residue is Bi; and (b) at positions3, 5, 8, 10, 11, 14, 17, 18, 24, 27, 32, 37, 38, 47, 48, 49, 52, 57, 58,61, 62, 63, 68, 69, 79, 80, 82, 83, 89, 92, 100, 101, 104, 119, 120,124, 125, 126, 128, 131, 143, and/or 144 the amino acid residue is B2;wherein B1 is an amino acid selected from the group consisting of A, I,L, M, F, W, Y, and V; and B2 is an amino acid selected from the groupconsisting of R, N, D, C, Q, E, G,H, K, P, S, andT.
 103. The isolated orrecombinant polypeptide of claim 52, wherein of the amino acid residuesin the amino acid sequence that correspond to the following positions,at least 80% conform to the following restrictions: (a) at positions 2,4, 15, 19, 26, 28, 51, 54, 86, 90, 91, 97, 103, 105, 106, 114, 129, 139,and/or 145 the amino acid residue is Z₁; (b) at positions 31, 45 and/or64 the amino acid residue is Z2; (c) at positions 8, 36 and/or 89 theamino acid residue is Z3 or Z6; (d) at positions 82, 92, 101 and/or 120the amino acid residue is Z4; (e) at positions 3, 11, 27 and/or 79 theamino acid residue is Z5; (f) at position 123 the amino acid residue isZ1 or Z2; (g) at positions 12, 33, 35, 39, 53, 59, 112, 132, 135, 140,and/or 146 the amino acid residue is Z1 or Z3; (h) at position 30 theamino acid residue is Z1 or Z4; (i) at position 6 the amino acid residueis Z1 or Z6; (j) at positions 81 and/or 113 the amino acid residue is Z2or Z3; (k) at positions 138 and/or 142 the amino acid residue is Z2 orZ4; (l) at positions 5, 17, 24, 57, 61, 124 and/or 126 the amino acidresidue is Z3, Z4 or Z6; (m) at position 104 the amino acid residue isZ3 or Z5; (o) at positions 38, 52, 62 and/or 69 the amino acid residueis Z1, Z3 or Z6; (p) at positions 14, 119 and/or 144 the amino acidresidue is Z1, Z2, Z4 or Z5; (q) at position 18 the amino acid residueis Z4, Z5 or Z6; (r) at positions 10, 32, 48, 63, 80 and/or 83 the aminoacid residue is Z5 or Z6; (s) at position 40 the amino acid residue isZ1, Z2 or Z3; (t) at positions 65 and/or 96 the amino acid residue isZ1, Z3, Z5 or Z6; (u) at positions 84 and/or 115 the amino acid residueis Z1, Z3 or Z4; (v) at position 93 the amino acid residue is Z2, Z3 orZ4; (w) at position 130 the amino acid residue is Z2, Z4 or Z6; (x) atpositions 47 and/or 58 the amino acid residue is Z3, Z4 or Z6; (y) atpositions 49, 68, 100 and/or 143 the amino acid residue is Z3, Z4 or Z5;(z) at position 131 the amino acid residue is Z3, Z5 or Z6; (aa) atpositions 125 and/or 128 the amino acid residue is Z4, Z5 or Z6; (ab) atposition 67 the amino acid residue is Z1, Z3, Z4 or Z5; (ac) at position60 the amino acid residue is Z1, Z4, Z5 or Z6; and (ad) at position 37the amino acid residue is Z3, Z4, Z5 or Z6; wherein Z1 is an amino acidselected from the group consisting of A, I, L, M, and V; Z2 is an aminoacid selected from the group consisting of F, W, and Y; Z3 is an aminoacid selected from the group consisting of N, Q, S, and T; Z4 is anamino acid selected from the group consisting of R, H, and K; Z5 is anamino acid selected from the group consisting of D and E; and Z6 is anamino acid selected from the group consisting of C, G, and P.
 104. Theisolated or recombinant polypeptide of claim 52, wherein of the aminoacid residues in the amino acid sequence that correspond to thefollowing positions, at least 90% conforn to the following restrictions:(a) at positions 1, 7, 9, 13, 20, 36, 42, 46, 50, 56, 64, 70, 72, 75,76, 78, 94, 98, 107, 110, 117, 118, 121, 141 and/or 144 the amino acidresidue is B1; and (b) at positions 16, 21, 22, 23, 25, 29, 34, 41, 43,44, 55, 66, 71, 73, 74, 77, 85, 87, 88, 95, 99, 102, 108, 109, 111, 116,122, 127, 133, 134, 136, 137 and/or 144 the amino acid residue is B2;wherein B 1 is an amino acid selected from the group consisting of A, I,L, M, F, W, Y, and V; and B2 is an amino acid selected from the groupconsisting of R, N, D, C, Q, E, G, H, K, P, S,andT.
 105. The isolated orrecombinant polypeptide of claim 52, wherein of the amino acid residuesin the amino acid sequence that correspond to the following positions,at least 90% conform to the following restrictions: (a) at positions 1,7, 9, 20, 36, 42, 50, 64, 72, 75, 76, 78, 94, 98, 110, 121, and/or 141the amino acid residue is Z1; (b) at positions 13, 46, 56, 64, 70, 107,117, and/or 118 the amino acid residue is Z2; (c) at positions 23, 36,55, 71, 77, 88, and/or 109 the amino acid residue is Z3; (d) atpositions 16, 21, 41, 73, 85, 99, and/or 111 the amino acid residue isZ4; (e) at positions 34 and/or 95 the amino acid residue is Z5; (f) atposition 22, 25, 29, 43, 44, 66, 74, 87, 102, 108, 116, 122, 127, 133,134, 136, and/or 137 the amino acid residue is Z6; wherein Z1 is anamino acid selected from the group consisting of A, I, L, M, and V; Z2is an amino acid selected from the group consisting of F, W, and Y; Z3is an amino acid selected from the group consisting of N, Q, S, and T;Z4 is an amino acid selected from the group consisting of R, H, and K;Z5 is an amino acid selected from the group consisting of D and E; andZ6 is an amino acid selected from the group consisting of C, G, and P.106. The isolated or recombinant polypeptide of claim 102, wherein ofthe amino acid residues in the amino acid sequence that correspond tothe following positions, at least 90% conform to the followingrestrictions: (a) at positions 1, 7, 9, 13, 20, 36, 42, 46, 50, 56, 64,70, 72, 75, 76, 78, 94, 98, 107, 110, 117, 118, 121, 141 and/or 144 theamino acid residue is B1; and (b) at positions 16, 21, 22, 23, 25, 29,34, 41, 43, 44, 55, 66, 71, 73, 74, 77, 85, 87, 88, 95, 99, 102, 108,109, 111, 116, 122, 127, 133, 134, 136, 137 and/or 144 the amino acidresidue is B2; wherein B1 is an amino acid selected from the groupconsisting of A, I, L, M, F, W, Y, and V; and B2 is an amino acidselected from the group consisting of R, N, D, C, Q, E, G, H, K, P, S,and T.
 107. The isolated or recombinant polypeptide of claim 103,wherein of the amino acid residues in the amino acid sequence thatcorrespond to the following positions, at least 90% conform to thefollowing restrictions: (a) at positions 1, 7, 9, 13, 20, 36, 42, 46,50, 56, 64, 70, 72, 75, 76, 78, 94, 98, 107, 110, 117, 118, 121, 141and/or 144 the amino acid residue is B1; and (b) at positions 16, 21,22, 23, 25, 29, 34, 41, 43, 44, 55, 66, 71, 73, 74, 77, 85, 87, 88, 95,99, 102, 108, 109, 111, 116, 122, 127, 133, 134, 136, 137 and/or 144 theamino acid residue is B2; wherein B 1 is an amino acid selected from thegroup consisting of A, I, L, M, F, W, Y, and V; and B2 is an amino acidselected from the group consisting of R, N, D, C, Q, E, G, H, K, P, S,and T.
 108. The isolated or recombinant polypeptide of claim 52, whereinof the amino acid residues in the amino acid sequence that correspond tothe following positions, at least 80% conform to the followingrestrictions: (a) at position 2 the amino acid residue is I or L; (b) atposition 3 the amino acid residue is E or D; (c) at position 4 the aminoacid residue is V, A or I; (d) at position 5 the amino acid residue isK, R or N; (e) at position 6 the amino acid residue is P or L; (f) atposition 8 the amino acid residue is N, S or T; (g) at position 10 theamino acid residue is E or G; (h) at position 11 the amino acid residueis D or E; (i) at position 12 the amino acid residue is T or A; (j) atposition 14 the amino acid residue is E, K or D; (k) at position 15 theamino acid residue is I or L; (l) at position 17 the amino acid residueis H or Q; (m) at position 18 the amino acid residue is R, C, K or E;(n) at position 19 the amino acid residue is I or V; (o) at position 24the amino acid residue is Q or R; (p) at position 26 the amino acidresidue is M, V, L or I; (q) at position 27 the amino acid residue is Eor D; (r) at position 28 the amino acid residue is A or V; (s) atposition 30 the amino acid residue is K, M, R or I; (t) at position 31the amino acid residue is Y or F; (u) at position 32 the amino acidresidue is E, G or D; (v) at position 33 the amino acid residue is T, Aor S; (w) at position 35 the amino acid residue is L, S or M; (x) atposition 37 the amino acid residue is R, G, E, Q or C; (y) at position38 the amino acid residue is G, S or D; (z) at position 39 the aminoacid residue is T, A or S; (aa) at position 40 the amino acid residue isF, L or S; (ab) at position 45 the amino acid residue is Y or F; (ac) atposition 47 the amino acid residue is R, Q or G; (ad) at position 48 theamino acid residue is G or D; (ae) at position 49 the amino acid residueis K, R, E or Q; (at) at position 51 the amino acid residue is I or V;(ag) at position 52 the amino acid residue is S, C or G; (ah) atposition 53 the amino acid residue is I, T or V; (ai) at position 54 theamino acid residue is A or V; (aj) at position 57 the amino acid residueis H or N; (ak) at position 58 the amino acid residue is Q, K, N, P orR; (al) at position 59 the amino acid residue is A or S; (am) atposition 60 the amino acid residue is E, K, G, V or D; (an) at position61 the amino acid residue is H or Q; (ao) at position 62 the amino acidresidue is P, L, S or T; (ap) at position 63 the amino acid residue isE, G or D; (aq) at position 65 the amino acid residue is E, D, V, P orQ; (ar) at position 67 the amino acid residue is Q, E, R, L, H or K;(as) at position 68 the amino acid residue is K, R, E, or N; (at) atposition 69 the amino acid residue is Q or P; (au) at position 79 theamino acid residue is E or D; (av) at position 80 the amino acid residueis G or E; (aw) at position 81 the amino acid residue is Y, H, N or F;(ax) at position 82 the amino acid residue is R or H; (ay) at position83 the amino acid residue is E, G or D; (az) at position 84 the aminoacid residue is Q, R or L; (ba) at position 86 the amino acid residue isA or V; (bb) at position 89 the amino acid residue is T, G or S; (bc) atposition 90 the amino acid residue is L or I; (bd) at position 91 theamino acid residue is I, L or V; (be) at position 92 the amino acidresidue is R or K; (bf) at position 93 the amino acid residue is H, Y orQ; (bg) at position 96 the amino acid residue is E, A or Q; (bh) atposition 97 the amino acid residue is L or I; (bi) at position 100 theamino acid residue is K, R, N or E; (bj) at position 101 the amino acidresidue is K or R; (bk) at position 103 the amino acid residue is A orV; (bl) at position 104 the amino acid residue is D or N; (bm) atposition 105 the amino acid residue is L, I or M; (bn) at position 106the amino acid residue is L or I; (bo) at position 112 the amino acidresidue is T, A or I; (bp) at position 113 the amino acid residue is S,T or F; (bq) at position 114 the amino acid residue is A or V; (br) atposition 115 the amino acid residue is S, R or A; (bs) at position 119the amino acid -residue is K, E or R; (bt) at position 120 the aminoacid residue is K or R; (bu) at position 123 the amino acid residue is For L; (bv) at position 124 the amino acid residue is S, C or R; (bw) atposition 125 the amino acid residue is E, K, G or D; (bx) at position126 the amino acid residue is Q or H; (by) at position 128 the aminoacid residue is E, D, G or K; (bz) at position 129 the amino acidresidue is V, I or A; (ca) at position 130 the amino acid residue is Y,H, F or C; (cb) at position 131 the amino acid residue is D, G, N or E;(cc) at position 132 the amino acid residue is I, T, A, M, V or L; (cd)at position 135 the amino acid residue is V, T, A or I; (ce) at position138 the amino acid residue is H or Y; (cf) at position 139 the aminoacid residue is I or V; (cg) at position 140 the amino acid residue isL, M or S; (ch) at position 142 the amino acid residue is Y or H; (ci)at position 143 the amino acid residue is K, R, T or E; (cj) at position144 the amino acid residue is K, E, W or R; (ck) at position 145 theamino acid residue is L or I; and (cl) at position 146 the amino acidresidue is T or A.
 109. The isolated or recombinant polypeptide of claim52, wherein of the amino acid residues in the amino acid sequence thatcorrespond to the following positions, at least 80% conform to thefollowing restrictions: (a) at position 9, 76, 94 and 110 the amino acidresidue is A; (b) at position 29 and 108 the arnino acid residue is C;(c) at position 34 the amino acid residue is D; (d) at position 95 theamino acid residue is E; (e) at position 56 the amino acid residue is F;(f) at position 43, 44, 66, 74, 87, 102, 116, 122, 127 and 136 the aminoacid residue is G; (g) at position 41 the amino acid residue is H; (h)at position 7 the amino acid residue is I; (i) at position 85 the aminoacid residue is K; (j) at position 20, 36, 42, 50, 72, 78, 98 and 121the amino acid residue is L; (k) at position 1, 75 and 141 the aminoacid residue is M; (l) at position 23, 64 and 109 the amino acid residueis N; (m) at position 22, 25, 133, 134 and 137 the amino acid residue isP; (n) at position 71 the amino acid residue is Q; (o) at position 16,21, 73, 99 and 111 the amino acid residue is R; (p) at position 55 and88 the amino acid residue is S; (q) at position 77 the amino acidresidue is T; (r) at position 107 the amino acid residue is W; and (s)at position 13, 46, 70, 117 and 118 the amino acid residue is Y. 110.The isolated or recombinant polypeptide of claim 108, wherein of theamino acid residues in the amino acid sequence that correspond to thefollowing positions, at least 90% conform to the following restrictions:(a) at positions 1, 7, 9, 13, 20, 36, 42, 46, 50, 56, 64, 70, 72, 75,76, 78, 94, 98, 107, 110, 117, 118, 121, 141 and/or 144 the amino acidresidue is BI; and (b) at positions 16, 21, 22, 23, 25, 29, 34, 41, 43,44, 55, 66, 71, 73, 74, 77, 85, 87, 88, 95, 99, 102, 108, 109, 111, 116,122, 127, 133,134, 136, 137 and/or 144 the amino acid residue is B2;wherein B1 is an amino acid selected from the group consisting of A, I,L, M, F, W, Y, and V; and B2 is an amino acid selected from the groupconsisting of R, N, D, C, Q, E, G, H, K, P, S, and T.
 111. The isolatedor recombinant polypeptide of claim 109, wherein of the amino acidresidues in the amino acid sequence that correspond to the followingpositions, at least 90% conform to the following restrictions: (a) atpositions 2, 4, 15, 19, 26, 28, 31, 45, 51, 54, 86, 90, 91, 97, 103,105, 106, 114, 123, 129, 139, 144 and/or 145 the amino acid residue isB1; and (b) at positions 3, 5, 8, 10, 11, 14, 17, 18, 24, 27, 32, 37,38, 47, 48, 49, 52, 57, 58, 61, 62, 63,68,69,79,80,82,83, 89,92, 100,101, 104, 119, 120, 124, 125, 126, 128, 131, 143, and/or 144 the aminoacid residue is B2; wherein B1 is an amino acid selected from the groupconsisting of A, I, L, M, F, W, Y, and V; and B2 is an amino acidselected from the group consisting of R, N, D, C, Q, E, G, H, K, P, S,and T.
 112. The isolated or recombinant polypeptide of claim 108,wherein of the amino acid residues in the amino acid sequence thatcorrespond to the following positions, at least 90% conform to thefollowing restrictions: (a) at positions 1, 7, 9, 20, 36, 42, 50, 64,72, 75, 76, 78, 94, 98, 110, 121, and/or 141 the amino acid residue isZ1; (b) at positions 13, 46, 56, 64, 70, 107, 117, and/or 118 the aminoacid residue is Z2; (c) at positions 23, 36, 55, 71, 77, 88, and/or 109the amino acid residue is Z3; (d) at positions 16, 21, 41, 73, 85, 99,and/or 111 the amino acid residue is Z4; (e) at positions 34 and/or 95the amino acid residue is Z5; (f) at position 22, 25, 29, 43, 44, 66,74, 87, 102, 108, 116, 122, 127, 133, 134, 136, and/or 137 the aminoacid residue is Z6; wherein Z₁ is an amino acid selected from the groupconsisting of A, I, L, M, and V; Z2 is an amino acid selected from thegroup consisting of F, W, and Y; Z3 is an amino acid selected from thegroup consisting of N, Q, S, and T; Z4 is an amino acid selected fromthe group consisting of R, H, and K; Z5 is an amino acid selected fromthe group consisting of D and E; and Z6 is an amino acid selected fromthe group consisting of C, G, and P.
 113. The isolated or recombinantpolypeptide of claim 109, wherein of the amino acid residues in theamino acid sequence that correspond to the following positions, at least80% conform to the following restrictions: (a) at positions 2, 4, 15,19, 26, 28, 51, 54, 86, 90, 91, 97, 103, 105, 106, 114, 129, 139, and/or145 the amino acid residue is Z1; (b) at positions 31, 45 and/oe 64 theamino acid residue is Z2; (c) at positions 8, 36 and/or 89 the aminoacid residue is Z3 or Z6; (d) at positions 82, 92, 101 and/or 120 theamino acid residue is Z4; (e) at positions 3, 11, 27 and/or 79 the aminoacid residue is Z5; (f) at position 123 the amino acid residue is Z1 orZ2; (g) at positions 12, 33, 35, 39, 53, 59, 112, 132, 135, 140, and/or146 the amino acid residue is Z1 or Z3; (h) at position 30 the aminoacid residue is Z1 or Z4; (i) at position 6 the amino acid residue is Z1or Z6; (j) at positions 81 and/or 113 the amino acid residue is Z2 orZ3; (k) at positions 138 and/or 142 the amino acid residue is Z2 or Z4;(l) at positions 5, 17, 24, 57, 61, 124 and/or 126 the amino acidresidue is Z3, Z4 or Z6; (m) at position 104 the amino acid residue isZ3 or Z5; (o) at positions 38, 52, 62 and/or 69 the amino acid residueis Z1, Z3 or Z6; (p) at positions 14, 119 and/or 144 the amino acidresidue is Z1, Z2, Z4 or Z5; (q) at position 18[E] the amino acidresidue is Z4, Z5 or Z6; (r) at positions 10, 32, 48, 63, 80 and/or 83the amino acid residue is Z5 or Z6; (s) at position 40 the amino acidresidue is Z 1, Z2 or Z3; (t) at positions 65 and/or 96 the amino acidresidue is Z1, Z3, Z5 or Z6; (u) at positions 84 and/or 115 the aminoacid residue is Z1, Z3 or Z4; (v) at position 93 the amino acid residueis Z2, Z3 or Z4; (w) at position 130 the amino acid residue is Z2, Z4 orZ6; (x) at positions 47 and/or 58 the amino acid residue is Z3, Z4 orZ6; (y) at positions 49, 68, 100 and/or 143 the amino acid residue isZ3, Z4 or Z5; (z) at position 131 the amino acid residue is Z3, Z5 orZ6; (aa) at positions 125 and/or 128 the amino acid residue is Z4, Z5 orZ6; (ab) at position 67 the amino acid residue is Z1, Z3, Z4 or Z5; (ac)at position 60 the amino acid residue is Z1, Z4, Z5 or Z6; and (ad) atposition 37 the amino acid residue is Z3, Z4, Z5 or Z6; wherein Z1 is anamino acid selected from the group consisting of A, I, L, M, and V; Z2is an amino acid selected from the group consisting of F, W, and Y; Z3is an amino acid selected from the group consisting of N, Q, S, and T;Z4 is an amino acid selected from the group consisting of R, H, and K;Z5 is an amino acid selected from the group consisting of D and E; andZ6 is an amino acid selected from the group consisting of C,G,and P.114. The isolated or recombinant polypeptide of claim 108, wherein ofthe amino acid residues in the amino acid sequence that correspond tothe following positions, at least 80% conforn to the followingrestrictions: (a) at position 9, 76, 94 and 110 the amino acid residueis A; (b) at position 29 and 108 the amino acid residue is C; (c) atposition 34 the amino acid residue is D; (d) at position 95 the aminoacid residue is E; (e) at position 56 the amino acid residue is F; (f)at position 43, 44, 66, 74, 87, 102, 116, 122, 127 and 136 the aminoacid residue is G; (g) at position 41 the amino acid residue is H; (h)at position 7 the amino acid residue is I; (i) at position 85 the aminoacid residue is K; (j) at position 20, 36, 42, 50, 72, 78, 98 and 121the amino acid residue is L; (k) at position 1, 75 and 141 the aminoacid residue is M; (l) at position 23, 64 and 109 the amino acid residueis N; (m) at position 22, 25, 133, 134 and 137 the amino acid residue isP; (n) at position 71 the amino acid residue is Q; (o) at position 16,21, 73, 99 and 111 the amino acid residue is R; (p) at position 55 and88 the amino acid residue is S; (q) at position 77 the amino acidresidue is T; (r) at position 107 the amino acid residue is W; and (s)at position 13, 46, 70, 117 and 118 the amino acid residue is Y. 115.The isolated or recombinant polypeptide of claim 25, wherein the aminoacid residue in the amino acid sequence that corresponds to position 28is V.
 116. The isolated or recombinant polypeptide of claim 25, whereinthe amino acid sequence is selected from the group consisting of SEQ IDNO:6-10, 263-514, 568-619, 621, 623, 625, 627, 629, 631, 633, 635, 637,639, 641, 643, 645, 647, 649, 651, 653, 655, 657, 659, 661, 663, 665,667, 669, 671, 673, 675, 677, 679, 681, 683, 685, 687,689,691,693,695,697, 699,701,703,705,707, 709,711,713,715,717,719,721,723, 725, 727, 729, 731, 733, 735, 737, 739, 741, 743, 745, 747,749, 751, 753, 755, 757, 759, 761, 763, 765, 767, 769, 771, 773, 775,777, 779, 781, 783, 785, 787, 789, 791, 793, 795, 797, 799, 801, 803,805, 807, 809, 811, and
 813. 117. A transgenic plant or transgenic plantexplant having an enhanced tolerance to glyphosate, wherein the plant orplant explant expresses a polypeptide withglyphosate-N-acetyltransferase activity and at least one polypeptideimparting glyphosate tolerance by an additional mechanism.
 118. Atransgenic plant or transgenic plant explant, wherein the plant or plantexplant expresses a polypeptide with glyphosate-N-acetyltransferaseactivity and at least one polypeptide imparting tolerance to anadditional herbicide.
 119. A transgenic plant or transgenic plantexplant having an enhanced tolerance to glyphosate, wherein the plant orplant explant expresses a polypeptide withglyphosate-N-acetyltransferase activity, at least one polypeptideimparting glyphosate tolerance by an additional mechanism, and at leastone polypeptide imparting tolerance to an additional herbicide.
 120. Atransgenic plant or transgenic plant explant having an enhancedtolerance to glyphosate, wherein the plant or plant explant expresses apolypeptide with glyphosate-N-acetyltransferase activity and at leastone of a polypeptide selected from the group consisting of aglyphosate-tolerant 5-enolpyruvylshikimate-3-phosphate synthase and aglyphosate-tolerant glyphosate oxido-reductase.
 121. A transgenic plantor transgenic plant explant, wherein the plant or plant explantexpresses a polypeptide with glyphosate-N-acetyltransferase activity andat least one polypeptide selected from the group consisting of a mutatedhydroxyphenylpyruvatedioxygenase, a sulfonamide-tolerant acetolactatesynthase, a sulfonamide-tolerant acetohydroxy acid synthase, animidazolinone-tolerant acetolactate synthase, an imidazolinone-tolerantacetohydroxy acid synthase, a phosphinothricin acetyl transferase and amutated protoporphyrinogen oxidase.
 122. A transgenic plant ortransgenic plant explant having an enhanced tolerance to glyphosate,wherein the plant or plant explant expresses a polypeptide withglyphosate-N-acetyltransferase activity, at least one of a firstpolypeptide selected from the group consisting of a glyphosate-tolerant5-enolpyruvylshikimate-3-phosphate synthase and a glyphosate-tolerantglyphosate oxido-reductase and at least one of a second polypeptideselected from the group consisting of a mutatedhydroxyphenylpyruvatedioxygenase, a sulfonamide-tolerant acetolactatesynthase, a sulfonamide-tolerant acetohydroxy acid synthase, animidazolinone-tolerant acetolactate synthase, an imidazolinone-tolerantacetohydroxy acid synthase, a phosphinothricin acetyl transferase and amutated protoporphyrinogen oxidase.
 123. A transgenic plant ortransgenic plant explant having an enhanced tolerance to glyphosate,wherein the plant or plant explant expresses a polypeptide withglyphosate-N-acetyltransferase activity and at least one polypeptideselected from the group consisting of a glyphosate-tolerant5-enolpyruvylshikimate-3-phosphate synthase, a glyphosate-tolerantglyphosate oxido-reductase, a mutated hydroxyphenylpyruvatedioxygenase,a sulfonamide-tolerant acetolactate synthase, a sulfonamide-tolerantacetohydroxy acid synthase, an imidazolinone-tolerant acetolactatesynthase, an imidazolinone-tolerant acetohydroxy acid synthase, aphosphinothricin acetyl transferase and a mutated protoporphyrinogenoxidase.
 124. A method for controlling weeds in a field containing acrop comprising: (a) planting the field with crop seeds or plants whichare transformed with a gene encoding a glyphosate-N-acetyltransferaseand at least one gene encoding a polypeptide imparting glyphosatetolerance by an additional mechanism; and (b) applying to the crop andweeds in the field an effective application of glyphosate sufficient toinhibit growth of the weeds in the field without significantly affectingthe crop.
 125. A method for preventing emergence of glyphosate resistantweeds in a field containing a crop comprising: (a) planting the fieldwith crop seeds or plants which are transformed with a gene encoding aglyphosate-N-acetyltransferase and at least one gene encoding apolypeptide imparting glyphosate tolerance by an additional mechanism;and (b) applying to the crop and weeds in the field an effectiveapplication of glyphosate.
 126. A method for selectively controllingweeds in a field containing a crop comprising: (a) planting the fieldwith crop seeds or plants which are transformed with a gene encoding aglyphosate-N-acetyltransferase and at least one gene encoding apolypeptide imparting tolerance to an additional herbicide, and; (b)applying to the crop and weeds in the field a simultaneous orchronologically staggered application of glyphosate and the additionalherbicide which is sufficient to inhibit growth of the weeds in thefield without significantly affecting the crop.
 127. A method forpreventing emergence of herbicide resistant weeds in a field containinga crop comprising: (a) planting the field with crop seeds or plantswhich are transformed with a gene encoding aglyphosate-N-acetyltransferase and at least one gene encoding apolypeptide imparting tolerance to an additional herbicide, and; (b)applying to the crop and weeds in the field a simultaneous orchronologically staggered application of glyphosate and the additionalherbicide.
 128. A method for selectively controlling weeds in a fieldcontaining a crop comprising: (a) planting the field with crop seeds orplants which are transformed with a gene encoding aglyphosate-N-acetyltransferase, at least one gene encoding a polypeptideimparting glyphosate tolerance by an additional mechanism and at leastone gene encoding a polypeptide imparting tolerance to an additionalherbicide, and; (b) applying to the crop and weeds in the field asimultaneous or chronologically staggered application of glyphosate andthe additional herbicide which is sufficient to inhibit growth of theweeds in the field without significantly affecting the crop.
 129. Amethod for preventing emergence of herbicide resistant weeds in a fieldcontaining a crop comprising: (a) planting the field with crop seeds orplants which are transformed with a gene encoding aglyphosate-N-acetyltransferase, at least one gene encoding a polypeptideimparting glyphosate tolerance by an additional mechanism and at leastone gene encoding a polypeptide imparting tolerance to an additionalherbicide, and; (b) applying to the crop and weeds in the field asimultaneous or chronologically staggered application of glyphosate andthe additional herbicide.
 130. The isolated or recombinantpolynucleotide of claim 1, wherein of the amino acid residues in theamino acid sequence that correspond to the following positions, one ormore conform to the following restrictions: (a) at position 75 the aminoacid is selected from the group consisting of B1, Z1, M or V; (b) atposition 58 the amino acid is selected from the group consisting of B2,Z3, Z4, Z6, K, P, Q or R; (c) at position 47 the amino acid is selectedfrom the group consisting of B2, Z4, Z6, R and G; (d) at position 45 theamino acid is selected from the group consisting of B 1, Z2, F or Y; (e)at position 91 the amino acid is selected from the group consisting ofB1, Z1, L, V or I; (f) at position 105 the amino acid is selected fromthe group consisting of B 1, Z1, I, M or L; (g) at position 129 theamino acid is selected from the group consisting of BI, Z1, I or V; and(h) at position 89 the amino acid is selected from the group consistingof B2, Z3, Z6, G, T or S; wherein B1 is an amino acid selected from thegroup consisting of A, I, L, M, F, W, Y, and V; B2 is an amino acidselected from the group consisting of R, N, D, C, Q, E, G, H, K, P, S,and T; Z1 is an amino acid selected from the group consisting of A, I,L, M, and V; Z2 is an amino acid selected from the group consisting ofF, W, and Y; Z3 is an amino acid selected from the group consisting ofN, Q, S, and T; Z4 is an amino acid selected from the group consistingof R, H, and K; Z5 is an amino acid selected from the group consistingof D and E; and Z6 is an amino acid selected from the group consistingof C, G, and P.
 131. The isolated or recombinant polypeptide of claim51, wherein of the amino acid residues in the amino acid sequence thatcorrespond to the following positions, one or more conform to thefollowing restrictions: (a) at position 75 the amino acid is selectedfrom the group consisting of B 1, Z1, M or V; (b) at position 58 theamino acid is selected from the group consisting of B2, Z3, Z4, Z6, K,P, Q or R; (c) at position 47 the amino acid is selected from the groupconsisting of B2, Z4, Z6, R and G; (d) at position 45 the amino acid isselected from the group consisting of B 1, Z2, F or Y; (e) at position91 the amino acid is selected from the group consisting of B 1, Z1, L, Vor I; (f) at position 105 the amino acid is selected from the groupconsisting of B 1, Z1, I, M or L; (g) at position 129 the amino acid isselected from the group consisting of B1, Z1, I or V; and (h) atposition 89 the amino acid is selected from the group consisting of B2,Z3, Z6, G, T or S; wherein B1 is an amino acid selected from the groupconsisting of A, I, L, M, F, W, Y, and V; B2 is an amino acid selectedfrom the group consisting of R, N, D, C, Q, E, G, H, K, P, S, and T; Z1is an amino acid selected from the group consisting of A, I, L, M, andV; Z2 is an amino acid selected from the group consisting of F, W, andY; Z3 is an amino acid selected from the group consisting of N, Q, S,and T; Z4 is an amino acid selected from the group consisting of R, H,and K; Z5 is an amino acid selected from the group consisting of D andE; and Z6 is an amino acid selected from the group consisting of C, G,and P.